MicromiRs

ABSTRACT

The present invention relates to very short heavily modified oligonucleotides which target and inhibit microRNAs in vivo, and their use in medicaments and pharmaceutical compositions.

RELATED APPLICATIONS

This application claims priority from four applications: U.S. 60/977,497 filed 4 Oct. 2007, U.S. 60/979,217 filed 11 Oct. 2007, U.S. 61/028,062, filed 12 Feb. 2008, and EP08104780, filed 17 Jul. 2008, all of which are hereby incorporated by reference. Furthermore we reference and incorporate by reference WO2007/112754 and WO2007/112753 which are earlier applications from the same applicants.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name: 2763_(—)0130005_sequence listing_ascii.txt; Size: 575,688 bytes; and Date of Creation: Dec. 13, 2012) filed with the application is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to very short oligonucleotides which target and inhibit microRNAs in vivo, and their use in medicaments and pharmaceutical compositions.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNAs) are an abundant class of short endogenous RNAs that act as post-transcriptional regulators of gene expression by base-pairing with their target mRNAs. They are processed from longer (ca 70-80 nt) hairpin-like precursors termed pre-miRNAs by the RNAse III enzyme Dicer. MicroRNAs assemble in ribonucleoprotein complexes termed miRNPs and recognize their target sites by antisense complementarity thereby mediating down-regulation of their target genes. Near-perfect or perfect complementarity between the miRNA and its target site results in target mRNA cleavage, whereas limited complementarity between the microRNA and the target site results in translational inhibition of the target gene.

A summary of the role of microRNAs in human diseases, and the inhibition of microRNAs using single stranded oligonucleotides is provided by WO2007/112754 and WO2007/112753, which are both hereby incorporated by reference in its entirety. WO2008046911, hereby incorporated by reference, provides microRNA sequences which are associated with cancer. Numerous microRNAs have been associated with disease phenotypes and it is therefore desirable to provide substances capable of modulating the availability of microRNAs in vivo. WO2007/112754 and WO2007/112753 disclose short single stranded oligonucleotides which are considered to form a strong duplex with their target miRNA. SEQ ID NOs 1-45 are examples of anti microRNA oligonucleotides as disclosed in WO2007/112754 and WO2007/112753.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that the use of very short oligonucleotides which target microRNAs and which have a high proportion of nucleotide analogue nucleotides, such as LNA nucleotides, are highly effective in alleviating the repression of RNAs, such as an mRNA, by the targeted microRNAs in vivo.

The present invention provides an oligomer a contiguous sequence of 7, 8, 9 or 10 nucleotide units in length, for use in reducing the effective amount of a microRNA target in a cell or an organism, wherein at least 70%, such as at least 80% of the nucleotide units of the oligomer are selected from the group consisting of LNA units and 2′ substituted nucleotide analogues.

The present invention provides an oligomer a contiguous sequence of 7, 8, 9 or 10 nucleotide units in length, for use in reducing the effective amount of a microRNA target in a cell or an organism, wherein at least 70% of the nucleotide units of the oligomer are selected from the group consisting of LNA units and 2′ substituted nucleotide analogues, and wherein at least 50%, such as at least 60%, such as at least 70% of the nucleotide units of the oligomer are LNA units.

The invention provides oligomers of between 7-10 nucleotides in length which comprises a contiguous nucleotide sequence of a total of between 7-10 nucleotides, such as 7, 8, 9, nucleotide units, wherein at least 50% of the nucleotide units of the oligomer are nucleotide analogues.

The invention further provides for an oligomer of between 7-10 nucleotides in length which comprises a contiguous nucleotide sequence of a total of between 7-10 nucleotides, such as 7, 8, 9, or 10, nucleotide units, wherein the nucleotide sequence is complementary to a corresponding nucleotide sequence found in mammalian or viral microRNA, and wherein at least 50% of the nucleotide units of the oligomer are nucleotide analogues.

The present invention provides olgiomers according to the invention as a medicament.

The present invention provides pharmaceutical compositions comprising the oligomer of the invention and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

The invention provides for a conjugate comprising an oligomer according to the invention, conjugated to at least one non-nucleotide or polynucleotide entity, such as a sterol, such as cholesterol.

The invention provides for the use of an oligomer or a conjugate according to the invention, for the manufacture of a medicament for the treatment of a disease or medical disorder associated with the presence or over-expression of a microRNA, such as one or more of the microRNAs referred to herein.

The invention provides for the treatment of a disease or medical disorder associated with the presence or overexpression of the microRNA, comprising the step of administering a composition (such as the pharmaceutical composition) comprising an oligomer or conjugate according to the invention to a patient suffering from or likely to suffer from said disease or medical disorder.

The invention provides for a method for reducing the effective amount of a microRNA target in a cell or an organism, comprising administering the oligomer of the invention, or a composition (such as a pharmaceutical composition) comprising the oligomer or conjugate according to the invention to the cell or organism.

The invention provides for a method for reducing the effective amount of a microRNA target in a cell or an organism, comprising administering the oligomer or conjugate or pharmaceutical composition according to the invention to the cell or organism.

The invention provides for a method for de-repression of a target mRNA (or one ore more RNAs) in a cell or an organism, comprising administering an oligomer or conjugate according to the invention, or a composition comprising said oligomer or conjugate, to said cell or organism.

The invention provides for the use of an oligomer or a conjugate according to the invention, for inhibiting the mircoRNA in a cell which comprises said microRNA, such as a human cell. The use may be in vivo or in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic presentation of the miR-21, miR-155 and miR-122 8-mer LNA-antimiRs (Compound 3205 (SEQ ID NO: 2); Compound 3207 (SEQ ID NO: 4); Compound 3208 (SEQ ID NO: 6), indicating the targeting positions with the fully LNA-modified and phosphorothiolated LNA-antimiR. Preferred hybridisation positions for 7-mer, 8-mer, 9-mer and 10-mer LNA oligonucleotides on the mature microRNA are also indicated.

FIG. 2. Assessment of miR-21 antagonism by Compound 3205 (SEQ ID NO: 2) and Compound 3204 (SEQ ID NO: 1) LNA-antimiRs in MCF-7 cells using a luciferase sensor assay. MCF-7 cells were co-transfected with luciferase sensor plasmids containing a perfect match target site for miR-21 or a mismatch target site (.mm2) and LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean of renilla/firefly ratios for three separate experiments (bars=s.e.m), were all have been normalized against 0 nM psiCHECK2 (=control).

FIG. 3. Assessment of miR-21 antagonism by Compound 3205 (SEQ ID NO: 2) and Compound 3204 (SEQ ID NO: 1) LNA-antimiRs in HeLa cells using a luciferase sensor assay. HeLa cells were co-transfected with luciferase sensor plasmids containing a perfect match target site for miR-21 (mir-21) or a mismatch target site (mm2) and LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean of renilla/firefly ratios for three separate experiments (bars=s.e.m), were all have been normalized against 0 nM psiCHECK2 (=control).

FIG. 4. Assessment of miR-155 antagonism by Compound 3206 (SEQ ID NO: 3) and Compound 3207 (SEQ ID NO: 4) LNA-antimiRs in LPS-treated mouse RAW cells using a luciferase sensor assay. RAW cells were co-transfected with miR-155 and the different LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean of renilla/firefly, were all have been normalized against 0 nM psiCHECK2.

FIG. 5. Assessment of miR-122 antagonism by Compound 3208 (SEQ ID NO: 6) and Compound 4 (SEQ ID NO: 5) LNA-antimiRs in HuH-7 cells using a luciferase sensor assay. HuH-7 cells were co-transfected with a miR-122 luciferase sensor containing a perfect match miR-122 target site and the different LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean of renilla/firefly ratios for three separate experiments (bars=s.e.m), where all have been normalized against 0 nM psiCHECK2 (=control).

FIG. 6. Schematic presentation of the miR-21 luciferase reporter constructs.

FIG. 7. Assessment of miR-21 antagonism by an 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) versus a 15-mer LNA-antimiR Compound 3204 (SEQ ID NO: 1) in PC3 cells using a luciferase reporter assay. PC3 cells were co-transfected with luciferase reporter plasmids containing a perfect match target site for miR-21 or a mismatch target site and LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) of three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=control). Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs. LNA nucleotides are indicated by ovals, and DNA residues are indicated by bars.

FIG. 8. Specificity assessment of miR-21 antagonism by an 8-mer LNA-antimiR in HeLa cells using a luciferase reporter assay. HeLa cells were co-transfected with luciferase reporter plasmids containing a perfect match or a mismatched target site for miR-21 and LNA-antimiRs Compound 3205 (SEQ ID NO: 2) or an 8-mer LNA mismatch control oligo Compound 3218 (SEQ ID NO: 16) at different concentrations. After 24 hours, cells were harvested and luciferase activity was measured. Shown are the mean values (bars=s.e.m) for three independent experiments where the Renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=control). Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs. Mismatches are indicated by filled ovals.

FIG. 9. Assessment of the shortest possible length of a fully LNA-modified LNA-antimiR that mediates effective antagonism of miR-21. HeLa cells were co-transfected with luciferase reporter plasmids containing a perfect match or a mismatch target site for miR-21 and the LNA-antimiRs at different concentrations (Compound 3209=6-mer (SEQ ID NO: 7) and Compound 3210=7-mer (SEQ ID NO: 8)). After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) for three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=control). Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs.

FIG. 10. Length assessment of fully LNA-substituted LNA-antimiRs antagonizing miR-21. HeLa cells were co-transfected with luciferase reporter plasmids containing a perfect match or a mismatch target site for miR-21 and LNA-antimiRs at different concentrations (Compound 3211=9-mer (SEQ ID NO: 9), Compound 3212=10-mer (SEQ ID NO: 10), Compound 3213=12-mer (SEQ ID NO: 11) and Compound 3214=14-mer (SEQ ID NO: 12)). After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) for three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=control). Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs.

FIG. 11. Determination of the most optimal position for an 8-mer LNA-antimiR within the miR target recognition sequence. HeLa cells were co-transfected with luciferase reporter plasmids containing a perfect match or a mismatch target site for miR-21 and the LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) for three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=control). Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs (Compound 3205 (SEQ ID NO: 2; Compound 3215 (SEQ ID NO: 13); Compound 3216 (SEQ ID NO: 14); Compound 3217 (SEQ ID NO: 15).

FIG. 12. Validation of interaction of the Pdcd-4-3′-UTR and miR-21 by the 8-mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR. HeLa cells were co-transfected with a luciferase reporter plasmid containing part of the 3′UTR of Pdcd4 gene and LNA-antimiRs at different concentrations (Compound 3205=8-mer, perfect match (SEQ ID NO: 2); Compound 3218=8-mer, mismatch (SEQ ID NO: 16); Compound 3204=15-mer, LNA/DNA mix (SEQ ID NO: 1); Compound 3220=15-mer, gapmer (SEQ ID NO: 18)). After 24 hours, cells were harvested and luciferase activity measured. Shown are renilla/firefly ratios that have been normalized against 0 nM. Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs.

FIG. 13. Comparison of an 8-mer LNA-antimiR (Compound 3207 (SEQ ID NO: 4)) with a 15-mer LNA-antimiR (Compound 3206 (SEQ ID NO: 3)) in antagonizing miR-155 in mouse RAW cells. Mouse RAW cells were co-transfected with luciferase reporter plasmids containing a perfect match for miR-155 and the different LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) of three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without miR-155 target site (=control). Shown is also a schematic presentation of the miR-155 sequence and the design and position of the LNA-antimiRs.

FIG. 14. Assessment of c/EBP□Assessment of c/EBP er LNAantimiR (Compound 3207 (SEQ ID NO: 4)) with a 15-mer LNA-antimiR (Compound 3206 (SEQ ID NO: 3)) in antagonizing miR-155 in mouse RAW cells. Mouse RAW cells were co-transfected with luciferase reporter plasmids containing a perfect match for miR-155 and the diffter 20 hours, cells were harvested and western blot analysis of protein extracts from RAW cells was performed. The different isoforms of c/EBP_(β) are indicated, and the ratios calculated on c/EBP_(β) LIP and beta-tubulin are shown below.

FIG. 15. Antagonism of miR-106b by a fully LNA-modified 8-mer (Compound 3221 (SEQ ID NO: 19)) LNA-antimiR or by a 15-mer mixmer (Compound 3228 (SEQ ID NO: 26)) antimiR. HeLa cells were co-transfected with luciferase reporter plasmids containing a perfect match for miR-106b and the different LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values of four replicates where the renilla/firefly ratios have been normalized against 0 nM empty vector without miRNA target site (=control). Shown is also a schematic presentation of the miR-106b sequence and the design and position of the LNA-antimiRs.

FIG. 16. Antagonism of miR-19b by a fully LNA-modified 8-mer (Compound 3222 (SEQ ID NO: 20)) LNA-antimiR and a 15-mer (Compound 3229 (SEQ ID NO: 27)) mixmer antimiR. HeLa cells were co-transfected with luciferase reporter plasmids containing a perfect match for miR-19a and the two LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values of four replicate experiments, where the renilla/firefly ratios have been normalized against 0 nM empty vector without a miR-19a target site (=control). Shown is also a schematic presentation of the miR-19a sequence and the design and position of the LNA-antimiRs.

FIG. 17. Schematic presentation showing the mature human miR-221 (SEQ ID NO: 432) and miR-222 (SEQ ID NO: 434) sequences. Shown in the square is the seed sequence (7-mer) that is conserved in both miRNA sequences.

FIG. 18. Targeting of a microRNA family using short, fully LNA-substituted LNA-antimiR. PC3 cells were co-transfected with luciferase reporter plasmids for miR-221 and miR-222 separately or together and with the different LNA-antimiRs at varying concentrations. When co-transfecting with the LNA-antimiRs (15-mers) Compound 3223 (SEQ ID NO: 21) (against miR-221) and Compound 3224 (SEQ ID NO: 22) (against miR-222), the total concentration was 2 nM (1 nM each), while transfecting the cells with Compound 3225 (SEQ ID NO: 23) (7-mer) the concentrations were 0, 1, 5, 10 or 25 nM. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) of three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without a miRNA target site (=control). Shown is also a schematic presentation of the miR-221/222 sequence and the design and position of the LNA-antimiRs.

FIG. 19. Assessment of p27 protein levels as a functional readout for antagonism of the miR-221/222 family by the 7-mer Compound 3225 (SEQ ID NO: 23) LNA-antimiR. PC3 cells were transfected with the 7-mer LNA-antimiR Compound 3225 (SEQ ID NO: 23) targeting both miR-221 and miR-222 at varying concentrations. After 24 hours, cells were harvested and protein levels were measured on a western blot. Shown are the ratios of p27/tubulin.

FIG. 20. Assessment of miR-21 antagonism by an 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) versus a 15-mer LNA-antimiR Compound 3204 (SEQ ID NO: 1) and an 8-mer with 2 mismatches Compound 3218 (SEQ ID NO: 16) in HepG2 cells using a luciferase reporter assay.

HepG2 cells were co-transfected with luciferase reporter plasmid containing a perfect match target site for miR-21 and LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) of three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=control). Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs.

FIG. 21. Validation of interaction of the Pdcd4 3′UTR and miR-21 by the 8-mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR versus the 15-mer Compound 3204 (SEQ ID NO: 1) and an 8-mer with two mismatches Compound 3218 (SEQ ID NO: 16).

Huh-7 cells were co-transfected with a luciferase reporter plasmid containing part of the 3′UTR of Pdcd4 gene, pre-miR-21 (10 nM) and LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) of three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=control). Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs.

FIG. 22. Antagonism of miR-21 by Compound 3205 (SEQ ID NO: 2) leads to increased levels of Pdcd4 protein levels.

HeLa cells were transfected with 5 nM LNA-antimiR Compound 3205 (SEQ ID NO: 2) (perfect match), or Compound 3219 (SEQ ID NO: 17) LNA scrambled (8mer) or Compound 3218 (SEQ ID NO: 16) (8-mer mismatch). Cells were harvested after 24 hours and subjected to Western blot with Pdcd4 antibody.

FIG. 23. ALT and AST levels in mice treated with Compound 3205 (SEQ ID NO: 2) (perfect match) or Compound 3218 (SEQ ID NO: 16) (mismatch control). Mice were sacrificed after 14 days and after receiving 25 mg/kg every other day.

FIG. 24. Assessment of PU.1 protein levels as a functional readout for miR-155 antagonism by short LNA-antimiR Compound 3207 (SEQ ID NO: 4).

THP-1 cells were co-transfected with pre-miR-155 (5 nmol) and different LNA oligonucleotides (5 nM) and 100 ng/ml LPS was added. After 24 hours, cells were harvested and western blot analysis of protein extracts from the THP-1 cells was performed. PU.1 and tubulin are indicated.

FIG. 25. Assessment of p27 protein levels as a functional readout for antagonism of the miR-221/222 family by the 7-mer Compound 3225 (SEQ ID NO: 23) LNA-antimiR.

PC3 cells were transfected with the 7-mer LNA-antimiR Compound 3225 (SEQ ID NO: 23) targeting both miR-221 and miR-222 and a LNA scrambled control at 5 and 25 nM. After 24 hours, cells were harvested and protein levels were measured on a western blot. Shown are the ratios of p27/tubulin.

FIG. 26. Knock-down of miR-221/222 by the 7-mer Compound 3225 (SEQ ID NO: 23) (perfect match) LNA-antimiR reduces colony formation in soft agar in PC3 cells.

PC3 cells were transfected with 25 nM of the 7-mer LNA-antimiR Compound 3225 (SEQ ID NO: 23) targeting both miR-221 and miR-222 or a 7-mer scrambled control Compound 3231 (SEQ ID NO: 28). After 24 hours, cells were harvested and seeded on soft agar. After 12 days, colonies were counted. One experiment has been done in triplicate.

FIG. 27. Overview of the human let-7 family, and of tested antagonists.

(upper) The sequences represent the mature miRNA for each member and the box depicts nucleotides 2-16, the positions typically antagonized by LNA-antimiRs (let-7a, SEQ ID NO: 96; let-7b, SEQ ID NO: 98; let-7c, SEQ ID NO: 100; let-7d, SEQ ID NO: 102; let-7e, SEQ ID NO: 104; let-7f, SEQ ID NO: 106; let-7i, SEQ ID NO: 111; miR-98, SEQ ID NO: 947). Columns to the right show the number of nucleotide differences compared to let-7a, within the seed (S: position 2-8), extended seed (ES; position 2-9), and the remaining sequence typically targeted by LNA-antimiRs (NE; position 9-16), respectively. Nucleotides with inverted colors are altered compared to let-7a. (lower) Summary of tested antagonists against the let-7 family, including information on design, length and perfectly complementary targets. All compounds are fully phoshorothiolated.

FIG. 28. Assessment of let-7 antagonism by six different LNA-antimiRs in Huh-7 cells using a luciferase sensor assay.

Huh-7 cells were co-transfected with luciferase sensor plasmids containing a partial HMGA2 3′UTR (with four let-7 binding sites), with or without let-7a precursor (grey and black bars, respectively), and with 6 different LNA-antimiRs at increasing concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean of renilla/firefly ratios for duplicate measurements and standard deviations for each assay. Within each LNA-antimiR group all ratios have been normalized to the average of wells containing no let-7a precursor (black bars).

FIG. 29. Luciferase results from Huh-7 cells transfected with the HMGA2 3′UTR sensor plasmid, LNA-antimiRs Compound 3226 (SEQ ID NO: 24) (left) and Compound 3227 (SEQ ID NO: 25) (right), and pre-miRs for let-7a (A), let-7d (B), let-7e (C), and let-7i (D). Grey bars indicate the target de-repression after pre-mis inclusion, whereas black control bars represent the equivalent level without pre-miR addition. Each ratio is based on quadruplicate measurements and have been normalized against the average of wells containing no precursor (black bars) within each treatment group.

FIG. 30. Luciferase results from HeLa cells transfected with the HMGA2 3′UTR sensor plasmid or control vector, and the LNA-antimiR Compound 3227 (SEQ ID NO: 25) at various concentrations. Each ratio is based on quadruplicate measurements normalized against untreated (0 nM) empty control vector (psi-CHECK-2; grey bars).

FIG. 31. Assessment of miR-21 antagonism by 8mer Compound 3205 (SEQ ID NO: 2) in HCT116 cells using a luciferase sensor assay. HCT116 cells were co-transfected with luciferase sensor plasmids containing a perfect match target site for miR-21 (grey bars) and LNA-antimiR and control oigonucleotides at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown is one typical example of two where the renilla/firefly ratios have been normalized against 0 nM empty vector (=black bars).

FIG. 32. Silencing of miR-21 by the 8-mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR reduces colony formation in soft agar in PC3 cells. PC3 cells were transfected with 25 nM of the 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) targeting miR-21. After 24 hours, cells were harvested and seeded on soft agar. After 12 days, colonies were counted. Shown is the mean of three separate experiments, each performed in triplicate, and normalised against 0 nM control (i.e. transfection but with no LNA). p=0.01898 for Compound 3205 (SEQ ID NO: 2).

FIG. 33. Knock-down of miR-21 by the 8-mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR reduces colony formation in soft agar in HepG2 cells. HepG2 cells were transfected with 25 nM of the 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) targeting miR-21. After 24 hours, cells were harvested and seeded on soft agar. After 17 days, colonies were counted. Shown is the mean of three replicates from one experiment (bars=SEM).

FIG. 34. Wound closure in the invasive human prostate cell line PC3 after treatment with Compound 3205 (SEQ ID NO: 2). (A) PC3 cells were transfected at day 3 with LNA-antimiR and control oligonucleotides at 25 nM, Compound 3205 (SEQ ID NO: 2) (8mer, perfect match) and Compound 3219 (SEQ ID NO: 17) (8mer, mismatch) and the following day a scratch was made. Pictures were taken after 24 hours in order to monitor the migration. (B) The area in each timepoint has been measured with the software program Image J and normalized against respective 0 h time-point.

FIG. 35. Length assessment of fully LNA-substituted LNA-antimiRs antagonizing miR-155. RAW cells were co-transfected with luciferase reporter plasmids containing a perfect match target site for miR-155 and with LNA-antimiR oligonucleotides at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) for three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=mock). Shown is also a schematic presentation of the miR sequence and the design and position of the LNA-antimiRs.

FIG. 36. Binding of 5′-FAM labeled LNA-antimiR-21 Compound 3205 (SEQ ID NO: 2) to mouse plasma protein.

(A) % unbound LNA-antimiR-21 compound as a function of oligonucleotide concentration in mouse plasma. (B) Concentration of unbound LNA-antimiR-21 Compound 3205 (SEQ ID NO: 2) as a function of Compound 3205 (SEQ ID NO: 2) concentration in mouse plasma.

FIG. 37. Quantification Ras protein levels by Western blot analysis. A. Gel image showing Ras and Tubulin (internal standard) protein in treated (anti-let-7; 8-mer) vs. untreated (saline) lung and kidney samples. B. Quantifications of Ras protein levels in the lung and kidney, respectively, of LNA-antimiR-treated mice (black bars), normalized against equivalent saline controls (grey bars), using tubulin as equal-loading control.

FIG. 38. Silencing of miR-21 by Compound 3205 (SEQ ID NO: 2) leads to increased levels of Pdcd4 protein levels in vivo. Mice were injected with saline or 25 mg/kg LNA-antimiR Compound 3205 (SEQ ID NO: 2) over 14 days every other day, with a total of 5 doses. Mice were sacrificed and protein was isolated from kidney and subjected to Western blot analysis with Pdcd4 antibody. A. Gel image showing Pdcd4 and Gapdh (internal standard) protein in treated (antimiR-21; 8-mer) vs. untreated (saline) kidney samples (M1, mouse 1; M2, mouse 2). B. Quantification of Pdcd4 protein levels in kidneys of LNA-antimiR-treated mice (dark grey bars), normalized against the average of equivalent saline controls (light grey bars), using Gapdh as loading control.

DETAILED DESCRIPTION OF THE INVENTION

Short oligonucleotides which incorporate LNA are known from the in vitro reagents area, (see for example WO2005/098029 and WO 2006/069584). However the molecules designed for diagnostic or reagent use are very different in design than those for in vivo or pharmaceutical use. For example, the terminal nucleotides of the reagent oligos are typically not LNA, but DNA, and the internucleoside linkages are typically other than phosphorothioate, the preferred linkage for use in the oligonucleotides of the present invention. The invention therefore provides for a novel class of oligonucleotides (referred to herein as oligomers) per se.

The following embodiments refer to certain embodiments of the oligomer of the invention, which may be used in a pharmaceutical composition. Aspects which refer to the oligomer may also refer to the contiguous nucleotide sequence, and vice versa.

The Oligomer

The oligomer of the invention is a single stranded oligonucleotide which comprises nucleotide analogues, such as LNA, which form part of, or the entire contiguous nucleotide sequence of the oligonucleotide. The nucleotide sequence of the oligomer consists of a contiguous nucleotide sequence.

The term “oligonucleotide” (or simply “oligo”), which is used interchangeably with the term “oligomer” refers, in the context of the present invention, to a molecule formed by covalent linkage of two or more nucleotides. When used in the context of the oligonucleotide of the invention (also referred to the single stranded oligonucleotide), the term “oligonucleotide” may have, in one embodiment, for example have between 7-10 nucleotides, such as in individual embodiments, 7, 8, 9, or 10.

The term ‘nucleotide’ refers to nucleotides, such as DNA and RNA, and nucleotide analogues. It should be recognised that, in some aspects, the term nucleobase may also be used to refer to a nucleotide which may be either naturally occurring or non-naturally occurring—in this respect the term nucleobase and nucleotide may be used interchangeably herein.

In some embodiments, the contiguous nucleotide sequence consists of 7 nucleotide analogues. In some embodiments, the contiguous nucleotide sequence consists of 8 nucleotide analogues. In some embodiments, the contiguous nucleotide sequence consists of 9 nucleotide analogues.

In one embodiment at least about 50% of the nucleotides of the oligomer are nucleotide analogues, such as at least about 55%, such as at least about 60%, or at least about 65% or at least about 70%, such as at least about 75%, such as at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95% or such as 100%. It will also be apparent that the oligonucleotide may comprise of a nucleotide sequence which consists of only nucleotide analogues. Suitably, the oligomer may comprise at least one LNA monomer, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA monomers. As described below, the contiguous nucleotide sequence may consist only of LNA units (including linkage groups, such as phosphorothioate linkages), or may consist of LNA and DNA units, or LNA and other nucleotide analogues. In some embodiments, the contiguous nucleotide sequence comprises either one or two DNA nucleotides, the remainder of the nucleotides being nucleotide analogues, such as LNA unit.

In some embodiments, the contiguous nucleotide sequence consists of 6 nucleotide analogues and a single DNA nucleotide. In some embodiments, the contiguous nucleotide consists of 7 nucleotide analogues and a single DNA nucleotide. In some embodiments, the contiguous nucleotide sequence consists of 8 nucleotide analogues and a single DNA nucleotide. In some embodiments, the contiguous nucleotide sequence consists of 9 nucleotide analogues and a single DNA nucleotide. In some embodiments, the contiguous nucleotide sequence consists of 7 nucleotide analogues and two DNA nucleotides. In some embodiments, the contiguous nucleotide sequence consists of 8 nucleotide analogues and two DNA nucleotides.

The oligomer may consist of the contiguous nucleotide sequence.

In a specially preferred embodiment, all the nucleotide analogues are LNA. In a further preferred embodiment, all nucleotides of the oligomer are LNA. In a further preferred embodiment, all nucleotides of the oligomer are LNA and all internucleoside linkage groups are phosphothioate.

Herein, the term “nitrogenous base” is intended to cover purines and pyrimidines, such as the DNA nucleobases A, C, T and G, the RNA nucleobases A, C, U and G, as well as non-DNA/RNA nucleobases, such as 5-methylcytosine (^(Me)C), isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 5-propyny-6-fluoroluracil, 5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine, 2,6-diaminopurine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanine and 2-chloro-6-aminopurine, in particular ^(Me)C. It will be understood that the actual selection of the non-DNA/RNA nucleobase will depend on the corresponding (or matching) nucleotide present in the microRNA strand which the oligonucleotide is intended to target. For example, in case the corresponding nucleotide is G it will normally be necessary to select a non-DNA/RNA nucleobase which is capable of establishing hydrogen bonds to G. In this specific case, where the corresponding nucleotide is G, a typical example of a preferred non-DNA/RNA nucleobase is ^(Me)C.

It should be recognised that the term in ‘one embodiment’ should not necessarily be limited to refer to one specific embodiment, but may refer to a feature which may be present in ‘some embodiments’, or even as a generic feature of the invention. Likewise, the use of the term ‘some emboidments’ may be used to describe a feature of one specific embodiment, or a collection of embodiments, or even as a generic feature of the invention.

The terms “corresponding to” and “corresponds to” refer to the comparison between the nucleotide sequence of the oligomer or contiguous nucleotide sequence (a first sequence) and the equivalent contiguous nucleotide sequence of a further sequence selected from either i) a sub-sequence of the reverse complement of the microRNA nucleic acid target (such as a microRNA target selected from SEQ ID NO:40 to SEQ ID NO:976, and/or ii) the sequence of 9-mer, 8-mer and 7-mer nucleotides provided herein in Table 1. Nucleotide analogues are compared directly to their equivalent or corresponding nucleotides. A first sequence which corresponds to a further sequence under i) or ii) typically is identical to that sequence over the length of the first sequence (such as the contiguous nucleotide sequence).

When referring to the length of a nucleotide molecule as referred to herein, the length corresponds to the number of monomer units, i.e. nucleotides, irrespective as to whether those monomer units are nucleotides or nucleotide analogues. With respect to nucleotides or nucleobases, the terms monomer and unit are used interchangeably herein.

It should be understood that when the term “about” is used in the context of specific values or ranges of values, the disclosure should be read as to include the specific value or range referred to.

As used herein, “hybridisation” means hydrogen bonding, which may be Watson-Crick, Hoogsteen, reversed Hoogsteen hydrogen bonding, etc., between complementary nucleoside or nucleotide bases. The four nucleobases commonly found in DNA are G, A, T and C of which G pairs with C, and A pairs with T. In RNA T is replaced with uracil (U), which then pairs with A. The chemical groups in the nucleobases that participate in standard duplex formation constitute the Watson-Crick face. Hoogsteen showed a couple of years later that the purine nucleobases (G and A) in addition to their Watson-Crick face have a Hoogsteen face that can be recognised from the outside of a duplex, and used to bind pyrimidine oligonucleotides via hydrogen bonding, thereby forming a triple helix structure.

In the context of the present invention “complementary” refers to the capacity for precise pairing between two nucleotides sequences with one another. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the corresponding position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The DNA or RNA strand are considered complementary to each other when a sufficient number of nucleotides in the oligonucleotide can form hydrogen bonds with corresponding nucleotides in the target DNA or RNA to enable the formation of a stable complex. To be stable in vitro or in vivo the sequence of an oligonucleotide need not be 100% complementary to its target microRNA. The terms “complementary” and “specifically hybridisable” thus imply that the oligonucleotide binds sufficiently strong and specific to the target molecule to provide the desired interference with the normal function of the target whilst leaving the function of non-target RNAs unaffected. However, in one preferred embodiment the term complementary shall mean 100% complementary or fully complementary.

In a preferred example the oligonucleotide of the invention is 100% complementary to a miRNA sequence, such as a human microRNA sequence, or one of the microRNA sequences referred to herein.

In a preferred example, the oligonucleotide of the invention comprises a contiguous sequence, which is 100% complementary to the seed region of the human microRNA sequence.

Preferably, the term “microRNA” or “miRNA”, in the context of the present invention, means an RNA oligonucleotide consisting of between 18 to 25 nucleotides in length. In functional terms miRNAs are typically regulatory endogenous RNA molecules.

The terms “target microRNA” or “target miRNA” refer to a microRNA with a biological role in human disease, e.g. an upregulated, oncogenic miRNA or a tumor suppressor miRNA in cancer, thereby being a target for therapeutic intervention of the disease in question.

The terms “target gene” or “target mRNA” refer to regulatory mRNA targets of microRNAs, in which said “target gene” or “target mRNA” is regulated post-transcriptionally by the microRNA based on near-perfect or perfect complementarity between the miRNA and its target site resulting in target mRNA cleavage; or limited complementarity, often conferred to complementarity between the so-called seed sequence (nucleotides 2-7 of the miRNA) and the target site resulting in translational inhibition of the target mRNA.

In the context of the present invention the oligonucleotide is single stranded, this refers to the situation where the oligonucleotide is in the absence of a complementary oligonucleotide—i.e. it is not a double stranded oligonucleotide complex, such as an siRNA. In one embodiment, the composition according of the invention does not comprise a further oligonucleotide which has a region of complementarity with the oligomer of 5 or more, such as 6, 7, 8, 9, or 10 consecutive nucleotides, such as eight or more.

Length

Surprisingly we have found that such short ‘antimiRs’ provide an improved specific inhibition of microRNAs in vivo, whilst retaining remarkable specificity for the microRNA target. A further benefit has been found to be the ability to inhibit several microRNAs simultaneously due to the conservation of homologous short sequences between microRNA species—such as the seed regions as described herein. According to the present invention, it has been found that it is particularly advantageous to have short oligonucleotides of 7, 8, 9, 10 nucleotides, such as 7, 8 or 9 nucleotides.

Sequences

The contiguous nucleotide sequence is complementary (such as 100% complementary—i.e. perfectly complementary) to a corresponding region of a mammalian, human or viral microRNA (miRNA) sequence, preferably a human or viral miRNA sequence.

The microRNA sequence may suitably be a mature microRNA. In some embodiments the microRNA may be a microRNA precursor.

The human microRNA sequence may be selected from SEQ ID NO:1 to SEQ ID NO: 558 as disclosed in WO2008/046911, which are all hereby and specifically incorporated by reference. As described in WO2008/046911, these microRNAs are associated with cancer.

The viral microRNA sequence may, in some embodiments, be selected from the group consisting of Herpes simplex virus 1, Kaposi sarcoma-associated herpesvirus, Epstein Barr virus and Human cytomegalovirus.

In one embodiment, the contiguous nucleotide sequence is complementary (such as 100% complementary) to a corresponding region of a miRNA sequence selected from the group of miRNAs listed in table 1. Table 1 provides 7-mer, 8-mer and 9-mer oligomers which target human and viral microRNAs published in miRBase (Release 12.0-http://microrna.sanger.ac.uk/sequences/).

In some embodiments, the oligomers according to the invention may consist of or comprise a contiguous nucleotide sequence which is complementary to a corresponding microRNA sequence selected from the group consisting of miR-1, miR-10b, miR-17-3p, miR-18, miR-19a, miR-19b, miR-20, miR-21, miR-34a, miR-93, miR-106a, miR-106b, miR-122, miR-133, miR-134, miR-138, miR-155, miR-192, miR-194, miR-221, miR-222, miR-375.

Therefore, in one embodiment, the miRNA (i.e target miRNA) is selected from the group consisting of miR-1, miR-10b, miR-17-3p, miR-18, miR-19a, miR-19b, miR-20, miR-21, miR-34a, miR-93, miR-106a, miR-106b, miR-122, miR-133, miR-134, miR-138, miR-155, miR-192, miR-194, miR-221, miR-222, and miR-375.

In one embodiment, the miRNA target is a member of the miR 17-92 cluster, such as miR 17, miR 106a, miR 106b, miR 18, miR 19a, miR 19b/1, miR 19b/2, miR20/93, miR92/1, miR92/2 and miR25.

In some embodiments the contiguous nucleotide sequence is complementary to a corresponding region of a microRNA (miRNA) sequence selected from the group consisting of miR-21, miR-155, miR-221, mir-222, and mir-122.

In some embodiments said miRNA is selected from the group consisting of miR-1, miR-10 miR-29, miR-125b, miR-126, miR-133, miR-141, miR-143, miR-200b, miR-206, miR-208, miR-302, miR-372, miR-373, miR-375, and miR-520c/e.

In some embodiments the contiguous nucleotide sequence is complementary to a corresponding region of a microRNA (miRNA) sequence present in the miR 17-92 cluster, such as a microRNA selected from the group consisting of miR-17-5p, miR-20a/b, miR-93, miR-106a/b, miR-18a/b, miR-19a/b, miR-25, miR-92a, miR-363.

In one embodiment, the miRNA (i.e target miRNA) is miR-21, such as hsa-miR-21 (SEQ ID NO: 410). In one embodiment, the miRNA (i.e target miRNA) is miR-122, such as hsa-miR-122 (SEQ ID NO: 150). In one embodiment, the miRNA (i.e target miRNA) is miR-19b, such as hsa-miR-19b (SEQ ID NO: 389). In one embodiment, the miRNA (i.e target miRNA) is miR-155, such as hsa-miR-155 (SEQ ID NO: 355). In one embodiment, the miRNA (i.e target miRNA) is miR-375, such as hsa-miR-375 (SEQ ID NO: 561). In one embodiment, the miRNA (i.e target miRNA) is miR-375, such as hsa-miR-106b (SEQ ID NO: 124).

Suitably, the contiguous nucleotide sequence may be complementary to a corresponding region of the microRNA, such as a hsa-miR selected from the group consisting of 19b (SEQ ID NO: 389), 21 (SEQ ID NO: 410), 122 (SEQ ID NO: 150), 155 (SEQ ID NO: 355) and 375 (SEQ ID NO: 561).

The Seed Region and Seedmers

The inventors have found that carefully designed short single stranded oligonucleotides comprising or consisting of nucleotide analogues, such as high affinity nucleotide analogues such as locked nucleic acid (LNA) units, show significant silencing of microRNAs, resulting in reduced microRNA levels. It was found that tight binding of said oligonucleotides to the so-called seed sequence, typically nucleotides 2 to 8 or 2 to 7, counting from the 5′ end, of the target microRNAs was important. Nucleotide 1 of the target microRNAs is a non-pairing base and is most likely hidden in a binding pocket in the Ago 2 protein. Whilst not wishing to be bound to a specific theory, the present inventors consider that by selecting the seed region sequences, particularly with oligonucleotides that comprise LNA, preferably LNA units in the region which is complementary to the seed region, the duplex between miRNA and oligonucleotide is particularly effective in targeting miRNAs, avoiding off target effects, and possibly providing a further feature which prevents RISC directed miRNA function.

The inventors have found that microRNA silencing is even more enhanced when LNA-modified single stranded oligonucleotides do not contain a nucleotide at the 3′ end corresponding to this non-paired nucleotide 1. It was further found that at least two LNA units in the 3′ end of the oligonucleotides according to the present invention made said oligonucleotides highly nuclease resistant.

In one embodiment, the first or second 3′ nucleotide of the oligomer corresponds to the second 5′ nucleotide of the microRNA sequence, and may be a nucleotide analogue, such as LNA.

In one embodiment, nucleotide units 1 to 6 (inclusive) of the oligomer as measured from the 3′ end the region of the oligomer are complementary to the microRNA seed region sequence, and may all be nucleotide analogues, such as LNA.

In one embodiment, nucleotide units 1 to 7 (inclusive) of the oligomer as measured from the 3′ end the region of the oligomer are complementary to the microRNA seed region sequence, and may all be nucleotide analogues, such as LNA.

In one embodiment, nucleotide units 2 to 7 (inclusive) of the oligomer as measured from the 3′ end the region of the oligomer are complementary to the microRNA seed region sequence, and may all be nucleotide analogues, such as LNA.

In one embodiment, the oligomer comprises at least one nucleotide analogue unit, such as at least one LNA unit, in a position which is within the region complementary to the miRNA seed region. The oligomer may, in one embodiment comprise at between one and 6 or between 1 and 7 nucleotide analogue units, such as between 1 and 6 and 1 and 7 LNA units, in a position which is within the region complementary to the miRNA seed region.

In one embodiment, the contiguous nucleotide sequence consists of or comprises a sequence which is complementary (such as 100% complementary) to the seed sequence of said microRNA.

In one embodiment, the contiguous nucleotide sequence consists of or comprises a sequence selected from any one of the seedmer sequences listed in table 1.

In one embodiment, the 3′ nucleotide of the seedmer forms the 3′ most nucleotide of the contiguous nucleotide sequence, wherein the contiguous nucleotide sequence may, optionally, comprise one or two further nucleotide 5′ to the seedmer sequence.

In one embodiment, the oligomer does not comprise a nucleotide which corresponds to the first nucleotide present in the microRNA sequence counted from the 5′ end.

In one embodiment, the oligonucleotide according to the invention does not comprise a nucleotide at the 3′ end that corresponds to the first 5′ end nucleotide of the target microRNA.

Nucleotide Analogues

According to the present invention, it has been found that it is particularly advantageous to have short oligonucleotides of 7, 8, 9, 10 nucleotides, such as 7, 8 or 9 nucleotides, wherein at least 50%, such as 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or such as 100% of the nucleotide units of the oligomer are (preferably high affinity) nucleotide analogues, such as a Locked Nucleic Acid (LNA) nucleotide unit.

In some embodiments, the oligonucleotide of the invention is 7, 8 or 9 nucleotides long, and comprises a contiguous nucleotide sequence which is complementary to a seed region of a human or viral microRNA, and wherein at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as 100% of the nucleotides are Locked Nucleic Acid (LNA) nucleotide units.

In such oligomers, in some embodiments, the linkage groups are other than phosphodiester linkages, such as are phosphorothioate linkages.

In one embodiment, all of the nucleotide units of the contiguous nucleotide sequence are LNA nucleotide units.

In one embodiment, the contiguous nucleotide sequence comprises or consists of 7, 8, 9 or 10, preferably contiguous, LNA nucleotide units.

In a further preferred embodiment, the oligonucleotide of the invention is 7, 8 or 9 nucleotides long, and comprises a contiguous nucleotide sequence which is complementary to a seed region of a human or viral microRNA, and wherein at least 80% of the nucleotides are LNA, and wherein at least 80%, such as 85%, such as 90%, such as 95%, such as 100% of the internucleotide bonds are phosphorothioate bonds. It will be recognised that the contiguous nucleotide sequence of the oligmer (a seedmer) may extend beyond the seed region.

In some embodiments, the oligonucleotide of the invention is 7 nucleotides long, which are all LNA.

In some embodiments, the oligonucleotide of the invention is 8 nucleotides long, of which up to 1 nucleotide may be other than LNA. In some embodiments, the oligonucleotide of the invention is 9 nucleotides long, of which up to 1 or 2 nucleotides may be other than LNA. In some embodiments, the oligonucleotide of the invention is 10 nucleotides long, of which 1, 2 or 3 nucleotides may be other than LNA. The nucleotides ‘other than LNA, may for example, be DNA, or a 2’ substituted nucleotide analogues.

High affinity nucleotide analogues are nucleotide analogues which result in oligonucleotides which has a higher thermal duplex stability with a complementary RNA nucleotide than the binding affinity of an equivalent DNA nucleotide. This may be determined by measuring the T_(m).

In some embodiments, the nucleotide analogue units present in the contiguous nucleotide sequence are selected, optionally independently, from the group consisting of 2′-O_alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, INA unit, and a 2′MOE RNA unit.

In some embodiments, the nucleotide analogue units present in the contiguous nucleotide sequence are selected, optionally independently, from the group consisting of 2′-O_alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA unit, and a 2′MOE RNA unit.

The term 2′ fluoro-DNA refers to a DNA analogue with a substitution to fluorine at the 2′ position (2′F). 2′ fluoro-DNA is a preferred form of 2′ fluoro-nucleotide.

In some embodiments, the oligomer comprises at least 4 nucleotide analogue units, such as at least 5 nucleotide analogue units, such as at least 6 nucleotide analogue units, such as at least 7 nucleotide analogue units, such as at least 8 nucleotide analogue units, such as at least 9 nucleotide analogue units, such as 10, nucleotide analogue units.

In one embodiment, the oligomer comprises at least 3 LNA units, such as at least 4 LNA units, such as at least 5 LNA units, such as at least 6 LNA units, such as at least 7 LNA units, such as at least 8 LNA units, such as at least 9 LNA units, such as 10 LNA.

In one embodiment wherein at least one of the nucleotide analogues, such as LNA units, is either cytosine or guanine, such as between 1-10 of the of the nucleotide analogues, such as LNA units, is either cytosine or guanine, such as 2, 3, 4, 5, 6, 7, 8, or 9 of the of the nucleotide analogues, such as LNA units, is either cytosine or guanine.

In one embodiment at least two of the nucleotide analogues such as LNA units are either cytosine or guanine. In one embodiment at least three of the nucleotide analogues such as LNA units are either cytosine or guanine. In one embodiment at least four of the nucleotide analogues such as LNA units are either cytosine or guanine. In one embodiment at least five of the nucleotide analogues such as LNA units are either cytosine or guanine. In one embodiment at least six of the nucleotide analogues such as LNA units are either cytosine or guanine. In one embodiment at least seven of the nucleotide analogues such as LNA units are either cytosine or guanine. In one embodiment at least eight of the nucleotide analogues such as LNA units are either cytosine or guanine.

In a preferred embodiment the nucleotide analogues have a higher thermal duplex stability for a complementary RNA nucleotide than the binding affinity of an equivalent DNA nucleotide to said complementary RNA nucleotide.

In one embodiment, the nucleotide analogues confer enhanced serum stability to the single stranded oligonucleotide.

Whilst the specific SEQ IDs in the sequence listing and table 1 refer to oligomers of LNA monomers with phosphorothioate (PS) backbone, it will be recognised that the invention also encompasses the use of other nucleotide analogues and/or linkages, either as an alternative to, or in combination with LNA. As such, the sequence of nucleotides (bases) shown in the sequence listings may be of LNA such as LNA/PS, LNA or may be oligomers containing alternative backbone chemistry, such as sugar/linkage chemistry, whilst retaining the same base sequence (A, T, C or G).

Whilst it is envisaged that other nucleotide analogues, such as 2′-MOE RNA or 2′-fluoro nucleotides may be useful in the oligomers according to the invention, it is preferred that the oligomers have a high proportion, such as at least 50%, LNA. nucleotides. The nucleotide analogue may be a DNA analogue such as a DNA analogue where the 2′-H group is substituted with a substitution other than —OH(RNA) e.g. by substitution with —O—CH₃, —O—CH₂—CH₂—O—CH₃, —O—CH₂—CH₂—CH₂—NH₂, —O—CH₂—CH₂—CH₂—OH or —F. The nucleotide analogue may be a RNA analogues such as a RNA analogue which have been modified in its 2′-OH group, e.g. by substitution with a group other than —H (DNA), for example —O—CH₃, —O—CH₂—CH₂—O—CH₃, —O—CH₂—CH₂—CH₂—NH₂, —O—CH₂—CH₂—CH₂—OH or —F. In one embodiment the nucleotide analogue is “ENA”.

LNA

When used in the present context, the terms “LNA unit”, “LNA monomer”, “LNA residue”, “locked nucleic acid unit”, “locked nucleic acid monomer” or “locked nucleic acid residue”, refer to a bicyclic nucleoside analogue. LNA units are described in inter alia WO 99/14226, WO 00/56746, WO 00/56748, WO 01/25248, WO 02/28875, WO 03/006475 and WO 03/095467. The LNA unit may also be defined with respect to its chemical formula. Thus, an “LNA unit”, as used herein, has the chemical structure shown in Scheme 1 below:

wherein

-   -   X is selected from the group consisting of O, S and NR^(H),         where R^(H) is H or C₁₋₄-alkyl; Y is (—CH₂)_(r), where r is an         integer of 1-4; and B is a nitrogenous base.         In a preferred embodiment of the invention, r is 1 or 2, in         particular 1, i.e. a preferred LNA unit has the chemical         structure shown in Scheme 2 below:

wherein X and B are as defined above. In an interesting embodiment, the LNA units incorporated in the oligonucleotides of the invention are independently selected from the group consisting of thio-LNA units, amino-LNA units and oxy-LNA units. Thus, the thio-LNA unit may have the chemical structure shown in Scheme 3 below:

wherein B is as defined above. Preferably, the thio-LNA unit is in its beta-D-form, i.e. having the structure shown in 3A above. likewise, the amino-LNA unit may have the chemical structure shown in Scheme 4 below:

wherein B and R^(H) are as defined above. Preferably, the amino-LNA unit is in its beta-D-form, i.e. having the structure shown in 4A above. The oxy-LNA unit may have the chemical structure shown in Scheme 5 below:

wherein B is as defined above.

Preferably, the oxy-LNA unit is in its beta-D-form, i.e. having the structure shown in 5A above. As indicated above, B is a nitrogenous base which may be of natural or non-natural origin. Specific examples of nitrogenous bases include adenine (A), cytosine (C), 5-methylcytosine (^(Me)C), isocytosine, pseudoisocytosine, guanine (G), thymine (T), uracil (U), 5-bromouracil, 5-propynyluracil, 5-propyny-6,5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine, 2,6-diaminopurine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanine and 2-chloro-6-aminopurine.

The term “thio-LNA unit” refers to an LNA unit in which X in Scheme 1 is S. A thio-LNA unit can be in both the beta-D form and in the alpha-L form. Generally, the beta-D form of the thio-LNA unit is preferred. The beta-D-form and alpha-L-form of a thio-LNA unit are shown in Scheme 3 as compounds 3A and 3B, respectively.

The term “amino-LNA unit” refers to an LNA unit in which X in Scheme 1 is NH or NR^(H), where R^(H) is hydrogen or C₁₋₄-alkyl. An amino-LNA unit can be in both the beta-D form and in the alpha-L form. Generally, the beta-D form of the amino-LNA unit is preferred. The beta-D-form and alpha-L-form of an amino-LNA unit are shown in Scheme 4 as compounds 4A and 4B, respectively.

The term “oxy-LNA unit” refers to an LNA unit in which X in Scheme 1 is O. An Oxy-LNA unit can be in both the beta-D form and in the alpha-L form. Generally, the beta-D form of the oxy-LNA unit is preferred. The beta-D form and the alpha-L form of an oxy-LNA unit are shown in Scheme 5 as compounds 5A and 5B, respectively.

In the present context, the term “C₁₋₆-alkyl” is intended to mean a linear or branched saturated hydrocarbon chain wherein the longest chains has from one to six carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl and hexyl. A branched hydrocarbon chain is intended to mean a C₁₋₆-alkyl substituted at any carbon with a hydrocarbon chain.

In the present context, the term “C₁₋₄-alkyl” is intended to mean a linear or branched saturated hydrocarbon chain wherein the longest chains has from one to four carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. A branched hydrocarbon chain is intended to mean a C₁₋₄-alkyl substituted at any carbon with a hydrocarbon chain.

When used herein the term “C₁₋₆-alkoxy” is intended to mean C₁₋₆-alkyl-oxy, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy, neopentoxy and hexoxy.

In the present context, the term “C₂₋₆-alkenyl” is intended to mean a linear or branched hydrocarbon group having from two to six carbon atoms and containing one or more double bonds. Illustrative examples of C₂₋₆-alkenyl groups include allyl, homo-allyl, vinyl, crotyl, butenyl, butadienyl, pentenyl, pentadienyl, hexenyl and hexadienyl. The position of the unsaturation (the double bond) may be at any position along the carbon chain.

In the present context the term “C₂₋₆-alkynyl” is intended to mean linear or branched hydrocarbon groups containing from two to six carbon atoms and containing one or more triple bonds. Illustrative examples of C₂₋₆-alkynyl groups include acetylene, propynyl, butynyl, pentynyl and hexynyl. The position of unsaturation (the triple bond) may be at any position along the carbon chain. More than one bond may be unsaturated such that the “C₂₋₆-alkynyl” is a di-yne or enedi-yne as is known to the person skilled in the art.

When referring to substituting a DNA unit by its corresponding LNA unit in the context of the present invention, the term “corresponding LNA unit” is intended to mean that the DNA unit has been replaced by an LNA unit containing the same nitrogenous base as the DNA unit that it has replaced, e.g. the corresponding LNA unit of a DNA unit containing the nitrogenous base A also contains the nitrogenous base A. The exception is that when a DNA unit contains the base C, the corresponding LNA unit may contain the base C or the base ^(Me)C, preferably ^(Me)C.

Herein, the term “non-LNA unit” refers to a nucleoside different from an LNA-unit, i.e. the term “non-LNA unit” includes a DNA unit as well as an RNA unit. A preferred non-LNA unit is a DNA unit.

The terms “unit”, “residue” and “monomer” are used interchangeably herein.

The term “at least one” encompasses an integer larger than or equal to 1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and so forth.

The terms “a” and “an” as used about a nucleotide, an agent, an LNA unit, etc., is intended to mean one or more. In particular, the expression “a component (such as a nucleotide, an agent, an LNA unit, or the like) selected from the group consisting of . . . ” is intended to mean that one or more of the cited components may be selected. Thus, expressions like “a component selected from the group consisting of A, B and C” is intended to include all combinations of A, B and C, i.e. A, B, C, A+B, A+C, B+C and A+B+C.

Internucleoside Linkages

The term “internucleoside linkage group” is intended to mean a group capable of covalently coupling together two nucleotides, such as between DNA units, between DNA units and nucleotide analogues, between two non-LNA units, between a non-LNA unit and an LNA unit, and between two LNA units, etc. Examples include phosphate, phosphodiester groups and phosphorothioate groups.

In some embodiments, at least one of, such as all of the internucleoside linkage in the oligomer is phosphodiester. However for in vivo use, phosphorothioate linkages may be preferred.

Typical internucleoside linkage groups in oligonucleotides are phosphate groups, but these may be replaced by internucleoside linkage groups differing from phosphate. In a further interesting embodiment of the invention, the oligonucleotide of the invention is modified in its internucleoside linkage group structure, i.e. the modified oligonucleotide comprises an internucleoside linkage group which differs from phosphate. Accordingly, in a preferred embodiment, the oligonucleotide according to the present invention comprises at least one internucleoside linkage group which differs from phosphate.

Specific examples of internucleoside linkage groups which differ from phosphate

(—O—P(O)₂—O—) include —O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—, —O—P(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—, O—PO(OCH₃)—O—, —O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—, —O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—, —NR^(H)—CO—NR^(H)—, —O—CO—O—, —O—CO—NR^(H)—, —NR^(H)—CO—CH₂—, —O—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—, —CO—NR^(H)—CH₂—, —CH₂—NR^(H)—CO—, —O—CH₂—CH₂—S—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—SO₂—CH₂—, —CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—CO—, —CH₂—NCH₃—O—CH₂—, where R^(H) is hydrogen or C₁₋₄-alkyl.

When the internucleoside linkage group is modified, the internucleoside linkage group is preferably a phosphorothioate group (—O—P(O,S)—O—). In a preferred embodiment, all internucleoside linkage groups of the oligonucleotides according to the present invention are phosphorothioate.

The internucleoside linkage may be selected form the group consisting of: —O—P(O)₂—O—, —O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—, —O—P(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—, O—PO(OCH₃)—O—, —O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—, —O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—, —NR^(H)—CO—NR^(H)—, and/or the internucleoside linkage may be selected form the group consisting of: —O—CO—O—, —O—CO—NR^(H)—, —NR^(H)—CO—CH₂—, —O—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—, —CO—NR^(H)—CH₂—, —CH₂—NR^(H)—CO—, —O—CH₂—CH₂—S—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—SO₂—CH₂—, —CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—CO—, —CH₂—NCH₃—O—CH₂—, where R^(H) is selected from hydrogen and C₁₋₄-alkyl. Suitably, in some embodiments, sulphur (S) containing internucleoside linkages as provided above may be preferred. The internucleoside linkages may be independently selected, or all be the same, such as phosphorothioate linkages.

In one embodiment, at least 75%, such as 80% or 85% or 90% or 95% or all of the internucleoside linkages present between the nucleotide units of the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

Micromir Oligonucleotides Targeting More than One microRNA

In one embodiment, the contiguous nucleotide sequence is complementary to the corresponding sequence of at least two miRNA sequences such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA sequence. The use of a single universal base may allow a single oligomer of the invention to target two independent microRNAs which either one or both have a single mismatch in the region which corresponds to oligomer at the position where the universal nucleotide is positioned.

In one embodiment, the contiguous nucleotide sequence consists of or comprises a sequence which is complementary to the sequence of at least two miRNA seed region sequences such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA seed region sequences.

In one embodiment, the contiguous nucleotide sequence is complementary to the corresponding region of both miR-221 and miR-222.

In one embodiment, the contiguous nucleotide sequence is complementary to the corresponding region of more than one member of the miR-17-92 cluster—such as two or more or all of miR-17-5p, miR-20a/b, miR-93, miR-106a/b; or two or more or all of miR-25, miR-92a and miR-363.

In one embodiment, the contiguous nucleotide sequence consists of or comprises a sequence that is complementary to 5′GCTACAT3′.

Oligomer Design

In one embodiment, the first nucleotide of the oligomer according to the invention, counting from the 3′ end, is a nucleotide analogue, such as an LNA unit. In one embodiment, which may be the same or different, the last nucleotide of the oligomer according to the invention, counting from the 3′ end, is a nucleotide analogue, such as an LNA unit.

In one embodiment, the second nucleotide of the oligomer according to the invention, counting from the 3′ end, is a nucleotide analogue, such as an LNA unit.

In one embodiment, the ninth and/or the tenth nucleotide of the oligomer according to the invention, counting from the 3′ end, is a nucleotide analogue, such as an LNA unit.

In one embodiment, the ninth nucleotide of the oligomer according to the invention, counting from the 3′ end is a nucleotide analogue, such as an LNA unit.

In one embodiment, the tenth nucleotide of the oligomer according to the invention, counting from the 3′ end is a nucleotide analogue, such as an LNA unit.

In one embodiment, both the ninth and the tenth nucleotide of the oligomer according to the invention, calculated from the 3′ end is a nucleotide analogue, such as an LNA unit.

In one embodiment, the oligomer according to the invention does not comprise a region of more than 3 consecutive DNA nucleotide units. In one embodiment, the oligomer according to the invention does not comprise a region of more than 2 consecutive DNA nucleotide units.

In one embodiment, the oligomer comprises at least a region consisting of at least two consecutive nucleotide analogue units, such as at least two consecutive LNA units. In one embodiment, the oligomer comprises at least a region consisting of at least three consecutive nucleotide analogue units, such as at least three consecutive LNA units.

Other Patterns of Nucleotide Analogues such as LNA in the Oligomer

Whilst it is envisaged that oligomers containing at least 6 LNA, such as at least 7 nucleotide units may be preferable, the discovery that such short oligomers are highly effective at targeting microRNAs in vivo can be used to prepare shorter oligomers of the invention which comprise other nucleotide analogues, such as high affinity nucleotide analogues. Indeed, the combination of LNA with other high affinity nucleotide analogues are considered as part of the present invention.

Modification of nucleotides in positions 1 to 2, counting from the 3′ end. The nucleotide at positions 1 and/or 2 may be a nucleotide analogue, such as a high affinity nucleotide analogue, such as LNA, or a nucleotide analogue selected from the group consisting of 2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, 2′-MOE-RNA unit, LNA unit, PNA unit, HNA unit, INA unit. The two 3′ nucleotide may therefore be

Xx, xX, XX or xx, wherein: In one embodiment X is LNA and x is DNA or another nucleotide analogue, such as as a 2′ substituted nucleotide analogue selected from the group consisting of 2′-O_alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA, and a 2′MOE RNA unit. Said non-LNA unit (x) may therefore be 2′MOE RNA or 2′-fluoro-DNA. Alternatively X is a nucleotide analogue, and x is DNA.

The above modification at the 2 3′ terminal nucleotides may be combined with modification of nucleotides in positions 3-8 counting from the 3′ end, as described below. In this respect nucleotides designated as X and x may be the same throughout the oligomer. It will be noted that when the oligomer is only 7 nucleotides in length the 8^(th) nucleotide counting from the 3′ end should be discarded. In the following embodiments which refer to the modification of nucleotides in positions 3 to 8, counting from the 3′ end, the LNA units, in one embodiment, may be replaced with other nucleotide analogues, such as those referred to herein. “X” may, therefore be selected from the group consisting of 2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, 2′-MOE-RNA unit, LNA unit, PNA unit, HNA unit, INA unit. “x” is preferably DNA or RNA, most preferably DNA. However, it is preferred that X is LNA.

In one embodiment of the invention, the oligonucleotides of the invention are modified in positions 3 to 8, counting from the 3′ end. The design of this sequence may be defined by the number of non-LNA units present or by the number of LNA units present. In a preferred embodiment of the former, at least one, such as one, of the nucleotides in positions three to eight, counting from the 3′ end, is a non-LNA unit. In another embodiment, at least two, such as two, of the nucleotides in positions three to eight, counting from the 3′ end, are non-LNA units. In yet another embodiment, at least three, such as three, of the nucleotides in positions three to eight, counting from the 3′ end, are non-LNA units. In still another embodiment, at least four, such as four, of the nucleotides in positions three to eight, counting from the 3′ end, are non-LNA units. In a further embodiment, at least five, such as five, of the nucleotides in positions three to eight, counting from the 3′ end, are non-LNA units. In yet a further embodiment, all six nucleotides in positions three to eight, counting from the 3′ end, are non-LNA units.

Alternatively defined, in an embodiment, the oligonucleotide according to the present invention comprises at least three LNA units in positions three to eight, counting from the 3′ end. In an embodiment thereof, the oligonucleotide according to the present invention comprises three LNA units in positions three to eight, counting from the 3′ end. The substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end, may be selected from the group consisting of XXXxxx, xXXXxx, xxXXXx, xxxXXX, XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit. In a preferred embodiment, the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end, is selected from the group consisting of XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit. In a more preferred embodiment, the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end, is selected from the group consisting of xXXxXx, xXXxxX, xxXXxX, xXxXXx, xXxxXX, xxXxXX and xXxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit. In an embodiment, the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end, is xXxXxX or XxXxXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit. In an embodiment, the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end, is xXxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.

In a further embodiment, the oligonucleotide according to the present invention comprises at least four LNA units in positions three to eight, counting from the 3′ end. In an embodiment thereof, the oligonucleotide according to the present invention comprises four LNA units in positions three to eight, counting from the 3′ end. The substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end, may be selected from the group consisting of xxXXXX, xXxXXX, xXXxXX, xXXXxX, xXXXXx, XxxXXX, XxXxXX, XxXXxX, XxXXXx, XXxxXX, XXxXxX, XXxXXx, XXXxxX, XXXxXx and XXXXxx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.

In yet a further embodiment, the oligonucleotide according to the present invention comprises at least five LNA units in positions three to eight, counting from the 3′ end. In an embodiment thereof, the oligonucleotide according to the present invention comprises five LNA units in positions three to eight, counting from the 3′ end. The substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end, may be selected from the group consisting of xXXXXX, XxXXXX, XXxXXX, XXXxXX, XXXXxX and XXXXXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.

Preferably, the oligonucleotide according to the present invention comprises one or two LNA units in positions three to eight, counting from the 3′ end. This is considered advantageous for the stability of the A-helix formed by the oligo:microRNA duplex, a duplex resembling an RNA:RNA duplex in structure.

In yet a further embodiment, the oligonucleotide according to the present invention comprises at least six LNA units in positions three to eight, counting from the 3′ end. In an embodiment thereof, the oligonucleotide according to the present invention comprises at from three to six LNA units in positions three to eight, counting from the 3′ end, and in addition from none to three other high affinity nucleotide analogues in the same region, such that the total amount of high affinity nucleotide analogues (including the LNA units) amount to six in the region from positions three to eight, counting from the 3′ end.

In some embodiments, such as when X is LNA, said non-LNA unit (x) is another nucleotide analogue unit, such as a 2′ substituted nucleotide analogue selected from the group consisting of 2′-O_alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA, and a 2′MOE RNA unit. Said non-LNA unit (x) may therefore be 2′MOE RNA or 2′-fluoro-DNA.

For oligomers which have 9 or 10 nucleotides, the nucleotide at positions 9 and/or 10 may be a nucleotide analogue, such as a high affinity nucleotide analogue, such as LNA, or a nucleotide analogue selected from the group consisting of 2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, 2′-MOE-RNA unit, LNA unit, PNA unit, HNA unit, INA unit. The two 5′ nucleotides may therefore be

Xx, xX, XX or xx, wherein: In one embodiment X is LNA and x is DNA or another nucleotide analogue, such as as a 2′ substituted nucleotide analogue selected from the group consisting of 2′-O_alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA, and a 2′MOE RNA unit. Said non-LNA unit (x) may therefore be 2′MOE RNA or 2′-fluoro-DNA. Alternatively X is a nucleotide analogue, and x is DNA.

The above modification at the 2 5′ terminal nucleotides may be combined with modification of nucleotides in positions 3-8 counting from the 3′ end, and/or the 2 3′ nucleotides as described above. In this respect nucleotides designated as X and x may be the same throughout the oligomer.

In a preferred embodiment of the invention, the oligonucleotide according to the present invention contains an LNA unit at the 5′ end. In another preferred embodiment, the oligonucleotide according to the present invention contains an LNA unit at the first two positions, counting from the 5′ end.

In one embodiment, the invention further provides for an oligomer as described in the context of the pharmaceutical composition of the invention, or for use in vivo in an organism, such as a medicament, wherein said oligomer (or contiguous nucleotide sequence) comprises either

i) at least one phosphorothioate linkage and/or

ii) at least one 3′ terminal LNA unit, and/or

iii) at least one 5′ terminal LNA unit.

The oligomer may therefore contain at least one phosphorothioate linkage, such as all linkages being phosphorothioates, and at least one 3′ terminal LNA unit, and at least one 5′ terminal LNA unit.

It is preferable for most therapeutic uses that the oligonucleotide is fully phosphorothiolated—an exception being for therapeutic oligonucleotides for use in the CNS, such as in the brain or spine where phosphorothioation can be toxic, and due to the absence of nucleases, phosphodiester bonds may be used, even between consecutive DNA units.

As referred to herein, other in one aspect of the oligonucleotide according to the invention is that the second 3′ nucleotide, and/or the 9^(th) and 10^(th) (from the 3′ end), if present, may also be LNA.

In one embodiment, the oligomer comprises at least five nucleotide analogue units, such as at least five LNA units, in positions which are complementary to the miRNA seed region.

In one embodiment, the nucleotide sequence of the oligomer which is complementary to the sequence of the microRNA seed region, is selected from the group consisting of (X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX, (X)XXXXxX and (X)XXXXXx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, (X) denotes an optional nucleotide analogue, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the oligomer comprises six or seven nucleotide analogue units, such as six or seven LNA units, in positions which are complementary to the miRNA seed region.

In one embodiment, the nucleotide sequence of the oligomer which is complementary to the sequence of the microRNA seed region, is selected from the group consisting of XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxXX, XXXXXxX and XXXXXXx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the two nucleotide motif at position 7 to 8, counting from the 3′ end of the oligomer is selected from the group consisting of xx, XX, xX and Xx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the two nucleotide motif at position 7 to 8, counting from the 3′ end of the oligomer is selected from the group consisting of XX, xX and Xx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the oligomer comprises at 12 nucleotides and wherein the two nucleotide motif at position 11 to 12, counting from the 3′ end of the oligomer is selected from the group consisting of xx, XX, xX and Xx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the oligomer comprises 12 nucleotides and wherein the two nucleotide motif at position 11 to 12, counting from the 3′ end of the oligomer is selected from the group consisting of XX, xX and Xx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit, such as a DNA unit.

In one embodiment, the oligomer comprises a nucleotide analogue unit, such as an LNA unit, at the 5′ end.

In one embodiment, the nucleotide analogue units, such as X, are independently selected form the group consisting of: 2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, 2′-MOE-RNA unit, LNA unit, PNA unit, HNA unit, INA unit.

In one embodiment, all the nucleotides of the oligomer of the invention are nucleotide analogue units.

In one embodiment, the nucleotide analogue units, such as X, are independently selected form the group consisting of: 2′-OMe-RNA units, 2′-fluoro-DNA units, and LNA units,

In one embodiment, the oligomer comprises said at least one LNA analogue unit and at least one further nucleotide analogue unit other than LNA.

In one embodiment, the non-LNA nucleotide analogue unit or units are independently selected from 2′-OMe RNA units and 2′-fluoro DNA units.

In one embodiment, the oligomer consists of at least one sequence XYX or YXY, wherein X is LNA and Y is either a 2′-OMe RNA unit and 2′-fluoro DNA unit.

In one embodiment, the sequence of nucleotides of the oligomer consists of alternative X and Y units.

In one embodiment, the oligomer comprises alternating LNA and DNA units (Xx) or (xX). In one embodiment, the oligomer comprises a motif of alternating LNA followed by 2 DNA units (Xxx), xXx or xxX.

In one embodiment, at least one of the DNA or non-LNA nucleotide analogue units are replaced with a LNA nucleotide in a position selected from the positions identified as LNA nucleotide units in any one of the embodiments referred to above. In one embodiment,“X” donates an LNA unit.

Further Designs for Oligomers of the Invention

Table 1 below provides non-limiting examples of short microRNA sequences that could advantageously be targeted with an oligonucleotide of the present invention.

The oligonucleotides according to the invention, such as those disclosed in table 1 may, in one embodiment, have a sequence of 7, 8, 9 or 10 LNA nucleotides 5′-3′ LLLLLLL(L)(L)(L)(L), or have a sequence of nucleotides selected form the group consisting of, the first 7, 8, 9 or 10 nucleotides of the following motifs:

LdLddL(L)(d)(d)(L)(d)(L)(d)(L)(L), LdLdLL(L)(d)(d)(L)(L)(L)(d)(L)(L), LMLMML(L)(M)(M)(L)(M)(L)(M)(L)(L), LMLMLL(L)(M)(M)(L)(L)(L)(M)(L)(L), LFLFFL(L)(F)(F)(L)(F)(L)(F)(L)(L), LFLFLL(L)(F)(F)(L)(L)(L)(F)(L)(L), and every third designs such as; LddLdd(L)(d)(d)(L)(d)(d)(L)(d)(d)(L)(d), dLddLd(d)(L)(d)(d)(L)(d)(d)(L)(d)(d)(L), ddLddL(d)(d)(L)(d)(d)(L)(d)(d)(L)(d)(d), LMMLMM(L)(M)(M)(L)(M)(M)(L)(M)(M)(L)(M), MLMMLM(M)(L)(M)(M)(L)(M)(M)(L)(M)(M)(L), MMLMML(M)(M)(L)(M)(M)(L)(M)(M)(L)(M)(M), LFFLFF(L)(F)(F)(L)(F)(F)(L)(F)(F)(L)(F), FLFFLF(F)(L)(F)(F)(L)(F)(F)(L)(F)(F)(L), FFLFFL(F)(F)(L)(F)(F)(L)(F)(F)(L)(F)(F), and dLdLdL(d)(L)(d)(L)(d)(L)(d)(L)(d)(L)(d) and an every second design, such as; LdLdLd(L)(d)(L)(d)(L)(d)(L)(d)(L)(d)(L), MLMLML(M)(L)(M)(L)(M)(L)(M)(L)(M)(L)(M), LMLMLM(L)(M)(L)(M)(L)(M)(L)(M)(L)(M)(L), FLFLFL(F)(L)(F)(L)(F)(L)(F)(L)(F)(L)(F), and LFLFLF(L)(F)(L)(F)(L)(F)(L)(F)(L)(F)(L); wherein L=LNA unit, d=DNA units, M=2′MOE RNA, F=2′Fluoro and residues in brackets are optional.

Pharmaceutical Composition and Medical Application

The invention provides for a pharmaceutical composition comprising the oligomer according to the invention, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

The invention further provides for the use of an oligonucleotide according to the invention, such as those which may form part of the pharmaceutical composition, for the manufacture of a medicament for the treatment of a disease or medical disorder associated with the presence or over-expression (upregulation) of the microRNA.

The invention further provides for a method for the treatment of a disease or medical disorder associated with the presence or over-expression of the microRNA, comprising the step of administering a composition (such as the pharmaceutical composition) according to the invention to a person in need of treatment.

The invention further provides for a method for reducing the effective amount of a miRNA in a cell or an organism, comprising administering a composition (such as the pharmaceutical composition) according to the invention or a oligomer according to the invention to the cell or the organism. Reducing the effective amount in this context refers to the reduction of functional miRNA present in the cell or organism. It is recognised that the preferred oligonucleotides according to the invention may not always significantly reduce the actual amount of miRNA in the cell or organism as they typically form very stable duplexes with their miRNA targets. The reduction of the effective amount of the miRNA in a cell may, in one embodiment, be measured by detecting the level of de-repression of the miRNA's target in the cell.

The invention further provides for a method for de-repression of a target mRNA of a miRNA in a cell or an organism, comprising administering a composition (such as the pharmaceutical composition) or a oligomer according to the invention to the cell or the organism.

The invention further provides for the use of a oligomer of between 7-10 such as 7, 8, 9, or 10 nucleotides in length, for the manufacture of a medicament for the treatment of a disease or medical disorder associated with the presence or over-expression of the microRNA.

In one embodiment the medical condition (or disease) is hepatitis C(HCV), and the miRNA is miR-122.

In one embodiment, the pharmaceutical composition according to the invention is for use in the treatment of a medical disorder or disease selected from the group consisting of: hepatitis C virus infection and hypercholesterolemia and related disorders, and cancers.

In one embodiment the medical disorder or disease is a CNS disease, such as a CNS disease where one or more microRNAs are known to be indicated.

In the context of hypercholesterolemia related disorders refers to diseases such as atherosclerosis or hyperlipidemia. Further examples of related diseases also include different types of HDL/LDL cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), acquired hyperlipidemia, statin-resistant hypercholesterolemia; coronary artery disease (CAD) coronary heart disease (CHD), atherosclerosis.

In one embodiment, the pharmaceutical composition according to the invention further comprises a second independent active ingredient that is an inhibitor of the VLDL assembly pathway, such as an ApoB inhibitor, or an MTP inhibitor (such as those disclosed in U.S. 60/977,497, hereby incorporated by reference).

The invention further provides for a method for the treatment of a disease or medical disorder associated with the presence or over-expression of the microRNA, comprising the step of administering a composition (such as the pharmaceutical composition) comprising a oligomer of between 7-10 such as 7, 8, 9, or 10 nucleotides in length, to a person in need of treatment.

The invention further provides for a method for reducing the effective amount of a miRNA target (i.e. ‘available’ miRNA) in a cell or an organism, comprising administering a composition (such as the pharmaceutical composition) comprising a oligomer of between 6 7-10 such as 7, 8, 9, or 10 nucleotides in length, to the cell or the organism.

It should be recognised that “reducing the effective amount” of one or more microRNAs in a cell or organism, refers to the inhibition of the microRNA function in the call or organism. The cell is preferably amammalain cell or a human cell which expresses the microRNA or microRNAs.

The invention further provides for a method for de-repression of a target mRNA of a miRNA in a cell or an organism, comprising a oligomer of 7-10 such as 7, 8, 9, or 10 nucleotides in length, or (or a composition comprising said oligonucleotide) to the cell or the organism.

As mentioned above, microRNAs are related to a number of diseases. Hence, a fourth aspect of the invention relates to the use of an oligonucleotide as defined herein for the manufacture of a medicament for the treatment of a disease associated with the expression of microRNAs selected from the group consisting of spinal muscular atrophy, Tourette's syndrome, hepatitis C, fragile X mental retardation, DiGeorge syndrome and cancer, such as in non limiting example, chronic lymphocytic leukemia, breast cancer, lung cancer and colon cancer, in particular cancer.

Methods of Synthesis

The invention further provides for a method for the synthesis of an oligomer targeted against a human microRNA, such as an oligomer described herein, said method comprising the steps of:

-   a. Optionally selecting a first nucleotide, counting from the 3′     end, which is a nucleotide analogue, such as an LNA nucleotide. -   b. Optionally selecting a second nucleotide, counting from the 3′     end, which is a nucleotide analogue, such as an LNA nucleotide. -   c. Selecting a region of the oligomer which corresponds to the miRNA     seed region, wherein said region is as defined herein. -   d. Selecting a seventh and optionally an eight nucleotide as defined     herein. -   e. Optionally selecting one or two further 5′ terminal of the     oligomer is as defined herein;

wherein the synthesis is performed by sequential synthesis of the regions defined in steps a-e, wherein said synthesis may be performed in either the 3′-5′ (a to f) or 5′-3′ (e to a) direction, and wherein said oligomer is complementary to a sequence of the miRNA target.

The invention further provides for a method for the preparation of an oligomer (such as an oligomer according to the invention), said method comprising the steps of a) comparing the sequences of two or more miRNA sequences to identify two or more miRNA sequences which comprise a common contiguous nucleotide sequence of at least 7 nucleotides in length, such as 7, 8, 9 or 10 nucleotides in length (i.e. a sequence found in both non-identical miRNAs), b) preparing an oligomer sequence which consists or comprises of a contiguous nucleotide sequence with is complementary to said common contiguous nucleotide sequence, wherein said oligomer is, as according to the oligomer of the invention. In a preferred example, the common contiguous nucleotide sequence consists or comprises of the seed region of each of said two or more miRNA sequences (which comprise a common contiguous nucleotide sequence of at least 6 nucleotides in length). In one embodiment, the seed regions of the two or more miRNAs are identical. Suitably the oligomer consists or comprises a seedmer sequence of 7 or 8 nucleotides in length which comprises of a sequence which is complementary to said two or more miRNAs. This method may be used in conjunction with step c of the above method.

The method for the synthesis of the oligomer according to the invention may be performed using standard solid phase oligonucleotide synthesis.

In one embodiment, the method for the synthesis of a oligomer targeted against a human microRNA, is performed in the 3′ to 5′ direction a-e.

A further aspect of the invention is a method to reduce the levels of target microRNA by contacting the target microRNA to an oligonucleotide as defined herein, wherein the oligonucleotide (i) is complementary to the target microRNA sequence (ii) does not contain a nucleotide at the 3′ end that corresponds to the first 5′ end nucleotide of the target microRNA.

Duplex Stability and T_(m)

In one embodiment, the oligomer of the invention is capable of forming a duplex with a complementary single stranded RNA nucleic acid molecule (typically of about the same length of said single stranded oligonucleotide) with phosphodiester internucleoside linkages, wherein the duplex has a T_(m) of between 30° C. and 70° C. or 80° C., such as between 30° C. and 60° C. of 70° C., or between 30° C. and 50° C. or 60° C. In one embodiment the T_(m) is at least 40° C. T_(m) may be determined by determining the T_(m) of the oligomer and a complementary RNA target in the following buffer conditions: 100 mM NaCl, 0.1 mM EDTA, 10 mM Na-phosphate, pH 7.0 (see examples for a detailed protocol). A high affinity analogue may be defined as an analogue which, when used in the oligomer of the invention, results in an increase in the T_(m) of the oligomer as compared to an identical oligomer which has contains only DNA bases.

Conjugates

In one embodiment, said oligomer is conjugated with one or more non-nucleotide (or poly-nucleotide) compounds.

In the context the term “conjugate” is intended to indicate a heterogenous molecule formed by the covalent attachment (“conjugation”) of the oligomer as described herein to one or more non-nucleotide, or non-polynucleotide moieties. Examples of non-nucleotide or non-polynucleotide moieties include macromolecular agents such as proteins, fatty acid chains, sugar residues, glycoproteins, polymers, or combinations thereof. Typically proteins may be antibodies for a target protein. Typical polymers may be polyethylene glycol.

Therefore, in various embodiments, the oligomer of the invention may comprise both a polynucleotide region which typically consists of a contiguous sequence of nucleotides, and a further non-nucleotide region. When referring to the oligomer of the invention consisting of a contiguous nucleotide sequence, the compound may comprise non-nucleotide components, such as a conjugate component.

In various embodiments of the invention the oligomeric compound is linked to ligands/conjugates, which may be used, e.g. to increase the cellular uptake of oligomeric compounds. WO2007/031091 provides suitable ligands and conjugates, which are hereby incorporated by reference.

The invention also provides for a conjugate comprising the compound according to the invention as herein described, and at least one non-nucleotide or non-polynucleotide moiety covalently attached to said compound. Therefore, in various embodiments where the compound of the invention consists of a specified nucleic acid or nucleotide sequence, as herein disclosed, the compound may also comprise at least one non-nucleotide or non-polynucleotide moiety (e.g. not comprising one or more nucleotides or nucleotide analogues) covalently attached to said compound.

Conjugation (to a conjugate moiety) may enhance the activity, cellular distribution or cellular uptake of the oligomer of the invention. Such moieties include, but are not limited to, antibodies, polypeptides, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g. Hexyl-s-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-o-hexadecyl-rac-glycero-3-h-phosphonate, a polyamine or a polyethylene glycol chain, an adamantane acetic acid, a palmityl moiety, an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

The oligomers of the invention may also be conjugated to active drug substances, for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

In certain embodiments the conjugated moiety is a sterol, such as cholesterol.

In various embodiments, the conjugated moiety comprises or consists of a positively charged polymer, such as a positively charged peptides of, for example between 1-50, such as 2-20 such as 3-10 amino acid residues in length, and/or polyalkylene oxide such as polyethylglycol(PEG) or polypropylene glycol—see WO 2008/034123, hereby incorporated by reference. Suitably the positively charged polymer, such as a polyalkylene oxide may be attached to the oligomer of the invention via a linker such as the releasable inker described in WO 2008/034123.

By way of example, the following conjugate moieties may be used in the conjugates of the invention:

Activated Oligomers

The term “activated oligomer,” as used herein, refers to an oligomer of the invention that is covalently linked (i.e., functionalized) to at least one functional moiety that permits covalent linkage of the oligomer to one or more conjugated moieties, i.e., moieties that are not themselves nucleic acids or monomers, to form the conjugates herein described. Typically, a functional moiety will comprise a chemical group that is capable of covalently bonding to the oligomer via, e.g., a 3′-hydroxyl group or the exocyclic NH₂ group of the adenine base, a spacer that is preferably hydrophilic and a terminal group that is capable of binding to a conjugated moiety (e.g., an amino, sulfhydryl or hydroxyl group). In some embodiments, this terminal group is not protected, e.g., is an NH₂ group. In other embodiments, the terminal group is protected, for example, by any suitable protecting group such as those described in “Protective Groups in Organic Synthesis” by Theodora W Greene and Peter G M Wuts, 3rd edition (John Wiley & Sons, 1999). Examples of suitable hydroxyl protecting groups include esters such as acetate ester, aralkyl groups such as benzyl, diphenylmethyl, or triphenylmethyl, and tetrahydropyranyl. Examples of suitable amino protecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl, triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acyl groups such as trichloroacetyl or trifluoroacetyl. In some embodiments, the functional moiety is self-cleaving. In other embodiments, the functional moiety is biodegradable. See e.g., U.S. Pat. No. 7,087,229, which is incorporated by reference herein in its entirety.

In some embodiments, oligomers of the invention are functionalized at the 5′ end in order to allow covalent attachment of the conjugated moiety to the 5′ end of the oligomer. In other embodiments, oligomers of the invention can be functionalized at the 3′ end. In still other embodiments, oligomers of the invention can be functionalized along the backbone or on the heterocyclic base moiety. In yet other embodiments, oligomers of the invention can be functionalized at more than one position independently selected from the 5′ end, the 3′ end, the backbone and the base.

In some embodiments, activated oligomers of the invention are synthesized by incorporating during the synthesis one or more monomers that is covalently attached to a functional moiety. In other embodiments, activated oligomers of the invention are synthesized with monomers that have not been functionalized, and the oligomer is functionalized upon completion of synthesis. In some embodiments, the oligomers are functionalized with a hindered ester containing an aminoalkyl linker, wherein the alkyl portion has the formula (CH₂)_(w), wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group is attached to the oligomer via an ester group (—O—C(O)—(CH₂)_(w)NH).

In other embodiments, the oligomers are functionalized with a hindered ester containing a (CH₂)_(w)-sulfhydryl (SH) linker, wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group attached to the oligomer via an ester group (—O—C(O)—(CH₂)_(w)SH).

In some embodiments, sulfhydryl-activated oligonucleotides are conjugated with polymer moieties such as polyethylene glycol or peptides (via formation of a disulfide bond).

Activated oligomers containing hindered esters as described above can be synthesized by any method known in the art, and in particular by methods disclosed in PCT Publication No. WO 2008/034122 and the examples therein, which is incorporated herein by reference in its entirety.

In still other embodiments, the oligomers of the invention are functionalized by introducing sulfhydryl, amino or hydroxyl groups into the oligomer by means of a functionalizing reagent substantially as described in U.S. Pat. Nos. 4,962,029 and 4,914,210, i.e., a substantially linear reagent having a phosphoramidite at one end linked through a hydrophilic spacer chain to the opposing end which comprises a protected or unprotected sulfhydryl, amino or hydroxyl group. Such reagents primarily react with hydroxyl groups of the oligomer. In some embodiments, such activated oligomers have a functionalizing reagent coupled to a 5′-hydroxyl group of the oligomer. In other embodiments, the activated oligomers have a functionalizing reagent coupled to a 3′-hydroxyl group. In still other embodiments, the activated oligomers of the invention have a functionalizing reagent coupled to a hydroxyl group on the backbone of the oligomer. In yet further embodiments, the oligomer of the invention is functionalized with more than one of the functionalizing reagents as described in U.S. Pat. Nos. 4,962,029 and 4,914,210, incorporated herein by reference in their entirety. Methods of synthesizing such functionalizing reagents and incorporating them into monomers or oligomers are disclosed in U.S. Pat. Nos. 4,962,029 and 4,914,210.

In some embodiments, the 5′-terminus of a solid-phase bound oligomer is functionalized with a dienyl phosphoramidite derivative, followed by conjugation of the deprotected oligomer with, e.g., an amino acid or peptide via a Diels-Alder cycloaddition reaction.

In various embodiments, the incorporation of monomers containing 2′-sugar modifications, such as a 2′-carbamate substituted sugar or a 2′-(O-pentyl-N-phthalimido)-deoxyribose sugar into the oligomer facilitates covalent attachment of conjugated moieties to the sugars of the oligomer. In other embodiments, an oligomer with an amino-containing linker at the 2′-position of one or more monomers is prepared using a reagent such as, for example, 5′-dimethoxytrityl-2′-O-(e-phthalimidylaminopentyl)-2′-deoxyadenosine-3′-N,N-diisopropyl-cyanoethoxy phosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters, 1991, 34, 7171.

In still further embodiments, the oligomers of the invention may have amine-containing functional moieties on the nucleotide, including on the N6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or 5 positions of cytosine. In various embodiments, such functionalization may be achieved by using a commercial reagent that is already functionalized in the oligomer synthesis.

Some functional moieties are commercially available, for example, heterobifunctional and homobifunctional linking moieties are available from the Pierce Co. (Rockford, Ill.). Other commercially available linking groups are 5′-Amino-Modifier C6 and 3′-Amino-Modifier reagents, both available from Glen Research Corporation (Sterling, Va.). 5′-Amino-Modifier C6 is also available from ABI (Applied Biosystems Inc., Foster City, Calif.) as Aminolink-2, and 3′-Amino-Modifier is also available from Clontech Laboratories Inc. (Palo Alto, Calif.).

Therapy and Pharmaceutical Compositions—Formulation and Administration

As explained initially, the oligonucleotides of the invention will constitute suitable drugs with improved properties. The design of a potent and safe drug requires the fine-tuning of various parameters such as affinity/specificity, stability in biological fluids, cellular uptake, mode of action, pharmacokinetic properties and toxicity.

Accordingly, in a further aspect the present invention relates to a pharmaceutical composition comprising an oligonucleotide according to the invention and a pharmaceutically acceptable diluent, carrier or adjuvant. Preferably said carrier is saline or buffered saline.

In a still further aspect the present invention relates to an oligonucleotide according to the present invention for use as a medicament.

As will be understood, dosing is dependent on severity and responsiveness of the disease state to be treated, and the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Optimum dosages may vary depending on the relative potency of individual oligonucleotides. Generally it can be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 μg to 1 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 10 years or by continuous infusion for hours up to several months. The repetition rates for dosing can be estimated based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state.

As indicated above, the invention also relates to a pharmaceutical composition, which comprises at least one oligonucleotide of the invention as an active ingredient. It should be understood that the pharmaceutical composition according to the invention optionally comprises a pharmaceutical carrier, and that the pharmaceutical composition optionally comprises further compounds, such as chemotherapeutic compounds, anti-inflammatory compounds, antiviral compounds and/or immuno-modulating compounds.

The oligonucleotides of the invention can be used “as is” or in form of a variety of pharmaceutically acceptable salts. As used herein, the term “pharmaceutically acceptable salts” refers to salts that retain the desired biological activity of the herein-identified oligonucleotides and exhibit minimal undesired toxicological effects. Non-limiting examples of such salts can be formed with organic amino acid and base addition salts formed with metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or with a cation formed from ammonia, N,N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, or ethylenediamine.

In one embodiment of the invention, the oligonucleotide may be in the form of a pro-drug. Oligonucleotides are by virtue negatively charged ions. Due to the lipophilic nature of cell membranes the cellular uptake of oligonucleotides are reduced compared to neutral or lipophilic equivalents. This polarity “hindrance” can be avoided by using the pro-drug approach (see e.g. Crooke, R. M. (1998) in Crooke, S. T. Antisense research and Application. Springer-Verlag, Berlin, Germany, vol. 131, pp. 103-140).

Pharmaceutically acceptable binding agents and adjuvants may comprise part of the formulated drug.

Examples of delivery methods for delivery of the therapeutic agents described herein, as well as details of pharmaceutical formulations, salts, may are well described elsewhere for example in U.S. provisional application 60/838,710 and 60/788,995, which are hereby incorporated by reference, and Danish applications, PA 2006 00615 which is also hereby incorporated by reference.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Delivery of drug to tumour tissue may be enhanced by carrier-mediated delivery including, but not limited to, cationic liposomes, cyclodextrins, porphyrin derivatives, branched chain dendrimers, polyethylenimine polymers, nanoparticles and microspheres (Dass C R. J Pharm Pharmacol 2002; 54(1):3-27). The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels and suppositories. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. The compounds of the invention may also be conjugated to active drug substances, for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

In another embodiment, compositions of the invention may contain one or more oligonucleotide compounds, targeted to a first microRNA and one or more additional oligonucleotide compounds targeted to a second microRNA target. Two or more combined compounds may be used together or sequentially.

The compounds disclosed herein are useful for a number of therapeutic applications as indicated above. In general, therapeutic methods of the invention include administration of a therapeutically effective amount of an oligonucleotide to a mammal, particularly a human. In a certain embodiment, the present invention provides pharmaceutical compositions containing (a) one or more compounds of the invention, and (b) one or more chemotherapeutic agents. When used with the compounds of the invention, such chemotherapeutic agents may be used individually, sequentially, or in combination with one or more other such chemotherapeutic agents or in combination with radiotherapy. All chemotherapeutic agents known to a person skilled in the art are here incorporated as combination treatments with compound according to the invention. Other active agents, such as anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, antiviral drugs, and immuno-modulating drugs may also be combined in compositions of the invention. Two or more combined compounds may be used together or sequentially.

Examples of therapeutic indications which may be treated by the pharmaceutical compositions of the invention:

microRNA Possible medical indications miR-1 Cardiac arythmia miR-21 Glioblastoma, breast cancer, hepatocellular carcinoma, colorectal cancer, sensitization of gliomas to cytotoxic drugs, cardiac hypertrophy miR-21, miR-200b Response to chemotherapy and regulation of and miR-141 cholangiocarcinoma growth miR-122 hypercholesterolemia, hepatitis C infection, hemochromatosis miR-19b lymphoma and other tumour types miR-26a Osteoblast differentiation of human stem cells miR-155 lymphoma, pancreatic tumor development, breast and lung cancer miR-203 Psoriasis miR-375 diabetes, metabolic disorders, glucose- induced insulin secretion from pancreatic endocrine cells miR-181 myoblast differentiation, auto immune disorders miR-10b Breast cancer cell invasion and metastasis miR-125b-1 Breast, lung, ovarian and cervical cancer miR-221 and 222 Prostate carcinoma, human thyroid papillary car, human hepatocellular carcinoma miRNA-372 and -373 testicular germ cell tumors. miR-142 B-cell leukemia miR-17-19b B-cell lymphomas, lung cancer, hepatocellular cluster carcinoma

Tumor suppressor gene tropomysin 1 (TPM1) mRNA has been indicated as a target of miR-21. Myotrophin (mtpn) mRNA has been indicated as a target of miR 375.

In an even further aspect, the present invention relates to the use of an oligonucleotide according to the invention for the manufacture of a medicament for the treatment of a disease selected from the group consisting of: atherosclerosis, hypercholesterolemia and hyperlipidemia; cancer, glioblastoma, breast cancer, lymphoma, lung cancer; diabetes, metabolic disorders; myoblast differentiation; immune disorders.

The invention further refers to oligonucleotides according to the invention for the use in the treatment of from a disease selected from the group consisting of: atherosclerosis, hypercholesterolemia and hyperlipidemia; cancer, glioblastoma, breast cancer, lymphoma, lung cancer; diabetes, metabolic disorders; myoblast differentiation; immune disorders.

The invention provides for a method of treating a subject suffering from a disease or condition selected from the group consisting of: atherosclerosis, hypercholesterolemia and hyperlipidemia; cancer, glioblastoma, breast cancer, lymphoma, lung cancer; diabetes, metabolic disorders; myoblast differentiation; immune disorders, the method comprising the step of administering an oligonucleotide or pharmaceutical composition of the invention to the subject in need thereof.

The invention further provides for a kit comprising a pharmaceutical composition according to the invention, and a second independent active ingredient that is an inhibitor of the VLDL assembly pathway, such as an ApoB inhibitor, or an MTP inhibitor.

Cancer

In an even further aspect, the present invention relates to the use of an oligonucleotide according to the invention for the manufacture of a medicament for the treatment of cancer. In another aspect, the present invention concerns a method for treatment of, or prophylaxis against, cancer, said method comprising administering an oligonucleotide of the invention or a pharmaceutical composition of the invention to a patient in need thereof.

Such cancers may include lymphoreticular neoplasia, lymphoblastic leukemia, brain tumors, gastric tumors, plasmacytomas, multiple myeloma, leukemia, connective tissue tumors, lymphomas, and solid tumors.

In the use of a compound of the invention for the manufacture of a medicament for the treatment of cancer, said cancer may suitably be in the form of a solid tumor. Analogously, in the method for treating cancer disclosed herein said cancer may suitably be in the form of a solid tumor.

Furthermore, said cancer is also suitably a carcinoma. The carcinoma is typically selected from the group consisting of malignant melanoma, basal cell carcinoma, ovarian carcinoma, breast carcinoma, non-small cell lung cancer, renal cell carcinoma, bladder carcinoma, recurrent superficial bladder cancer, stomach carcinoma, prostatic carcinoma, pancreatic carcinoma, lung carcinoma, cervical carcinoma, cervical dysplasia, laryngeal papillomatosis, colon carcinoma, colorectal carcinoma and carcinoid tumors. More typically, said carcinoma is selected from the group consisting of malignant melanoma, non-small cell lung cancer, breast carcinoma, colon carcinoma and renal cell carcinoma. The malignant melanoma is typically selected from the group consisting of superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral melagnoma, amelanotic melanoma and desmoplastic melanoma.

Alternatively, the cancer may suitably be a sarcoma. The sarcoma is typically in the form selected from the group consisting of osteosarcoma, Ewing's sarcoma, chondrosarcoma, malignant fibrous histiocytoma, fibrosarcoma and Kaposi's sarcoma.

Alternatively, the cancer may suitably be a glioma.

A further embodiment is directed to the use of an oligonucleotide according to the invention for the manufacture of a medicament for the treatment of cancer, wherein said medicament further comprises a chemotherapeutic agent selected from the group consisting of adrenocorticosteroids, such as prednisone, dexamethasone or decadron; altretamine (hexylen, hexamethylmelamine (HMM)); amifostine (ethyol); aminoglutethimide (cytadren); amsacrine (M-AMSA); anastrozole (arimidex); androgens, such as testosterone; asparaginase (elspar); bacillus calmette-gurin; bicalutamide (casodex); bleomycin (blenoxane); busulfan (myleran); carboplatin (paraplatin); carmustine (BCNU, BiCNU); chlorambucil (leukeran); chlorodeoxyadenosine (2-CDA, cladribine, leustatin); cisplatin (platinol); cytosine arabinoside (cytarabine); dacarbazine (DTIC); dactinomycin (actinomycin-D, cosmegen); daunorubicin (cerubidine); docetaxel (taxotere); doxorubicin (adriomycin); epirubicin; estramustine (emcyt); estrogens, such as diethylstilbestrol (DES); etopside (VP-16, VePesid, etopophos); fludarabine (fludara); flutamide (eulexin); 5-FUDR (floxuridine); 5-fluorouracil (5-FU); gemcitabine (gemzar); goserelin (zodalex); herceptin (trastuzumab); hydroxyurea (hydrea); idarubicin (idamycin); ifosfamide; IL-2 (proleukin, aldesleukin); interferon alpha (intron A, roferon A); irinotecan (camptosar); leuprolide (lupron); levamisole (ergamisole); lomustine (CCNU); mechlorathamine (mustargen, nitrogen mustard); melphalan (alkeran); mercaptopurine (purinethol, 6-MP); methotrexate (mexate); mitomycin-C (mutamucin); mitoxantrone (novantrone); octreotide (sandostatin); pentostatin (2-deoxycoformycin, nipent); plicamycin (mithramycin, mithracin); prorocarbazine (matulane); streptozocin; tamoxifin (nolvadex); taxol (paclitaxel); teniposide (vumon, VM-26); thiotepa; topotecan (hycamtin); tretinoin (vesanoid, all-trans retinoic acid); vinblastine (valban); vincristine (oncovin) and vinorelbine (navelbine). Suitably, the further chemotherapeutic agent is selected from taxanes such as Taxol, Paclitaxel or Docetaxel.

Similarly, the invention is further directed to the use of an oligonucleotide according to the invention for the manufacture of a medicament for the treatment of cancer, wherein said treatment further comprises the administration of a further chemotherapeutic agent selected from the group consisting of adrenocorticosteroids, such as prednisone, dexamethasone or decadron; altretamine (hexylen, hexamethylmelamine (HMM)); amifostine (ethyol); aminoglutethimide (cytadren); amsacrine (M-AMSA); anastrozole (arimidex); androgens, such as testosterone; asparaginase (elspar); bacillus calmette-gurin; bicalutamide (casodex); bleomycin (blenoxane); busulfan (myleran); carboplatin (paraplatin); carmustine (BCNU, BiCNU); chlorambucil (leukeran); chlorodeoxyadenosine (2-CDA, cladribine, leustatin); cisplatin (platinol); cytosine arabinoside (cytarabine); dacarbazine (DTIC); dactinomycin (actinomycin-D, cosmegen); daunorubicin (cerubidine); docetaxel (taxotere); doxorubicin (adriomycin); epirubicin; estramustine (emcyt); estrogens, such as diethylstilbestrol (DES); etopside (VP-16, VePesid, etopophos); fludarabine (fludara); flutamide (eulexin); 5-FUDR (floxuridine); 5-fluorouracil (5-FU); gemcitabine (gemzar); goserelin (zodalex); herceptin (trastuzumab); hydroxyurea (hydrea); idarubicin (idamycin); ifosfamide; IL-2 (proleukin, aldesleukin); interferon alpha (intron A, roferon A); irinotecan (camptosar); leuprolide (lupron); levamisole (ergamisole); lomustine (CCNU); mechlorathamine (mustargen, nitrogen mustard); melphalan (alkeran); mercaptopurine (purinethol, 6-MP); methotrexate (mexate); mitomycin-C (mutamucin); mitoxantrone (novantrone); octreotide (sandostatin); pentostatin (2-deoxycoformycin, nipent); plicamycin (mithramycin, mithracin); prorocarbazine (matulane); streptozocin; tamoxifin (nolvadex); taxol (paclitaxel); teniposide (vumon, VM-26); thiotepa; topotecan (hycamtin); tretinoin (vesanoid, all-trans retinoic acid); vinblastine (valban); vincristine (oncovin) and vinorelbine (navelbine). Suitably, said treatment further comprises the administration of a further chemotherapeutic agent selected from taxanes, such as Taxol, Paclitaxel or Docetaxel.

Alternatively stated, the invention is furthermore directed to a method for treating cancer, said method comprising administering an oligonucleotide of the invention or a pharmaceutical composition according to the invention to a patient in need thereof and further comprising the administration of a further chemotherapeutic agent. Said further administration may be such that the further chemotherapeutic agent is conjugated to the compound of the invention, is present in the pharmaceutical composition, or is administered in a separate formulation.

Infectious Diseases

It is contemplated that the compounds of the invention may be broadly applicable to a broad range of infectious diseases, such as diphtheria, tetanus, pertussis, polio, hepatitis B, hepatitis C, hemophilus influenza, measles, mumps, and rubella.

Hsa-miR122 (SEQ ID NO: 150) is indicated in hepatitis C infection and as such oligonucleotides according to the invention which target miR-122 may be used to treat Hepatitus C infection.

Accordingly, in yet another aspect the present invention relates the use of an oligonucleotide according to the invention for the manufacture of a medicament for the treatment of an infectious disease, as well as to a method for treating an infectious disease, said method comprising administering an oligonucleotide according to the invention or a pharmaceutical composition according to the invention to a patient in need thereof.

In a preferred embodiment, the invention provides for a combination treatment providing an anti miR-122 oligomer in combination with an inhibitor of VLDL assembly, such as an inhibitor of apoB, or of MTP.

Inflammatory Diseases

The inflammatory response is an essential mechanism of defense of the organism against the attack of infectious agents, and it is also implicated in the pathogenesis of many acute and chronic diseases, including autoimmune disorders. In spite of being needed to fight pathogens, the effects of an inflammatory burst can be devastating. It is therefore often necessary to restrict the symptomatology of inflammation with the use of anti-inflammatory drugs. Inflammation is a complex process normally triggered by tissue injury that includes activation of a large array of enzymes, the increase in vascular permeability and extravasation of blood fluids, cell migration and release of chemical mediators, all aimed to both destroy and repair the injured tissue.

In yet another aspect, the present invention relates to the use of an oligonucleotide according to the invention for the manufacture of a medicament for the treatment of an inflammatory disease, as well as to a method for treating an inflammatory disease, said method comprising administering an oligonucleotide according to the invention or a pharmaceutical composition according to the invention to a patient in need thereof.

In one preferred embodiment of the invention, the inflammatory disease is a rheumatic disease and/or a connective tissue diseases, such as rheumatoid arthritis, systemic lupus erythematous (SLE) or Lupus, scleroderma, polymyositis, inflammatory bowel disease, dermatomyositis, ulcerative colitis, Crohn's disease, vasculitis, psoriatic arthritis, exfoliative psoriatic dermatitis, pemphigus vulgaris and Sjorgren's syndrome, in particular inflammatory bowel disease and Crohn's disease.

Alternatively, the inflammatory disease may be a non-rheumatic inflammation, like bursitis, synovitis, capsulitis, tendinitis and/or other inflammatory lesions of traumatic and/or sportive origin.

Metabolic Diseases

A metabolic disease is a disorder caused by the accumulation of chemicals produced naturally in the body. These diseases are usually serious, some even life threatening. Others may slow physical development or cause mental retardation. Most infants with these disorders, at first, show no obvious signs of disease. Proper screening at birth can often discover these problems. With early diagnosis and treatment, metabolic diseases can often be managed effectively.

In yet another aspect, the present invention relates to the use of an oligonucleotide according to the invention or a conjugate thereof for the manufacture of a medicament for the treatment of a metabolic disease, as well as to a method for treating a metabolic disease, said method comprising administering an oligonucleotide according to the invention or a conjugate thereof, or a pharmaceutical composition according to the invention to a patient in need thereof.

In one preferred embodiment of the invention, the metabolic disease is selected from the group consisting of Amyloidosis, Biotimidase, OMIM (Online Mendelian Inheritance in Man), Crigler Najjar Syndrome, Diabetes, Fabry Support & Information Group, Fatty acid Oxidation Disorders, Galactosemia, Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency, Glutaric aciduria, International Organization of Glutaric Acidemia, Glutaric Acidemia Type I, Glutaric Acidemia, Type II, Glutaric Acidemia Type I, Glutaric Acidemia Type-II, F-HYPDRR-Familial Hypophosphatemia, Vitamin D Resistant Rickets, Krabbe Disease, Long chain 3 hydroxyacyl CoA dehydrogenase deficiency (LCHAD), Mannosidosis Group, Maple Syrup Urine Disease, Mitochondrial disorders, Mucopolysaccharidosis Syndromes: Niemann Pick, Organic acidemias, PKU, Pompe disease, Porphyria, Metabolic Syndrome, Hyperlipidemia and inherited lipid disorders, Trimethylaminuria: the fish malodor syndrome, and Urea cycle disorders.

Liver Disorders

In yet another aspect, the present invention relates to the use of an oligonucleotide according to the invention or a conjugate thereof for the manufacture of a medicament for the treatment of a liver disorder, as well as to a method for treating a liver disorder, said method comprising administering an oligonucleotide according to the invention or a conjugate thereof, or a pharmaceutical composition according to the invention to a patient in need thereof.

In one preferred embodiment of the invention, the liver disorder is selected from the group consisting of Biliary Atresia, Alagille Syndrome, Alpha-1 Antitrypsin, Tyrosinemia, Neonatal Hepatitis, and Wilson Disease.

Other Uses

The oligonucleotides of the present invention can be utilized for as research reagents for diagnostics, therapeutics and prophylaxis. In research, the oligonucleotide may be used to specifically inhibit the synthesis of target genes in cells and experimental animals thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention. In diagnostics the oligonucleotides may be used to detect and quantitate target expression in cell and tissues by Northern blotting, in-situ hybridisation or similar techniques. For therapeutics, an animal or a human, suspected of having a disease or disorder, which can be treated by modulating the expression of target is treated by administering the oligonucleotide compounds in accordance with this invention. Further provided are methods of treating an animal particular mouse and rat and treating a human, suspected of having or being prone to a disease or condition, associated with expression of target by administering a therapeutically or prophylactically effective amount of one or more of the oligonucleotide compounds or compositions of the invention.

Therapeutic Use of Oligonucleotides Targeting miR-122a

We have demonstrated that a LNA-antimiR, targeting miR-122a reduces plasma cholesterol levels. Therefore, another aspect of the invention is use of the above described oligonucleotides targeting miR-122a as medicine.

Still another aspect of the invention is use of the above described oligonucleotides targeting miR-122a for the preparation of a medicament for treatment of increased plasma cholesterol levels (or hypercholesterolemia and related disorders). The skilled man will appreciate that increased plasma cholesterol levels is undesirable as it increases the risk of various conditions, e.g. atherosclerosis.

Still another aspect of the invention is use of the above described oligonucleotides targeting miR-122a for upregulating the mRNA levels of Nrdg3, Aldo A, Bckdk or CD320.

Embodiments

The following embodiments of the present invention may be used in combination with the other embodiments described herein.

1. A pharmaceutical composition comprising an oligomer of between 6-12 nucleotides in length, wherein said oligomer comprises a contiguous nucleotide sequence of a total of between 6-12 nucleotides, such as 6, 7, 8, 9, 10, 11 or 12 nucleotide units, wherein at least 50% of the nucleobase units of the oligomer are high affinity nucleotide analogue units, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant. 2. The pharmaceutical composition according to embodiment 1, wherein the contiguous nucleotide sequence is complementary to a corresponding region of a mammalian, human or viral microRNA (miRNA) sequence. 3. The pharmaceutical composition according to embodiment 2, wherein the contiguous nucleotide sequence is complementary to a corresponding region of a miRNA sequence selected from the group of miRNAs listed in any one of tables 3, 4 or 5. 4. The pharmaceutical composition according to embodiment 2 or 3, wherein the contiguous nucleotide sequence consists of or comprises a sequence which is complementary to the seed sequence of said microRNA. 5. The pharmaceutical composition according to any one of embodiments 2-4, wherein the contiguous nucleotide sequence consists of or comprises a sequence selected from any one of the sequences listed in table 3 or 4. 6. The pharmaceutical composition according to embodiment 4 or 5, wherein the 3′ nucleobase of the seedmer forms the 3′ most nucleobase of the contiguous nucleotide sequence, wherein the contiguous nucleotide sequence may, optionally, comprise one or two further 5′ nucleobases. 7. The pharmaceutical composition according to any one of embodiments 1-6, wherein said contiguous nucleotide sequence does not comprise a nucleotide which corresponds to the first nucleotide present in the micro RNA sequence counted from the 5′ end. 8. The pharmaceutical composition according to any one of embodiments 1-7, wherein the contiguous nucleotide sequence is complementary to a corresponding nucleotide sequence present in a miRNA selected from those shown in table 3 or 4 or 5. 9. The pharmaceutical composition according to embodiment 8, wherein said miRNA is selected from the group consisting of miR-1, miR-10b, miR-17-3p, miR-18, miR-19a, miR-19b, miR-20, miR-21, miR-34a, miR-93, miR-106a, miR-106b, miR-122, miR-133, miR-134, miR-138, miR-155, miR-192, miR-194, miR-221, miR-222, and miR-375. 10. The pharmaceutical composition according to any one of embodiments 1-9, wherein at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or all of the nucleobase units of the contiguous nucleotide sequence are nucleotide analogue units. 11. The pharmaceutical composition according to embodiment 10, wherein the nucleotide analogue units are selected from the group consisting of 2′-O_alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, INA unit, and a 2′MOE RNA unit. 12. The pharmaceutical composition according to embodiment 10 or 11, wherein at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or all of the nucleobase units of the contiguous nucleotide sequence are Locked Nucleic Acid (LNA) nucleobase units. 13. The pharmaceutical composition according to embodiment 12, wherein all of the nucleobase units of the contiguous nucleotide sequence are LNA nucleobase units. 14. The pharmaceutical composition according to any one of embodiments 1-13, wherein the contiguous nucleotide sequence comprises or consists of 7, 8, 9 or 10, preferably contiguous, LNA nucleobase units. 15. The pharmaceutical composition according to any one of embodiments 1-14, wherein the oligomer consist of 7, 8, 9 or 10 contiguous nucleobase units and wherein at least 7 nucleobase units are nucleotide analogue units. 16. The pharmaceutical composition according to embodiment 15, wherein the nucleotide analogue units are Locked Nucleic Acid (LNA) nucleobase units. 17. The pharmaceutical composition according to embodiment 15, wherein the nucleotide analogue units in the molecule consists of a mixture of at least 50% LNA units and up to 50% other nucleotide analogue units. 18. The pharmaceutical composition according to any one of embodiments 1-17, wherein at least 75%, such as 80% or 85% or 90% or 95% or all of the internucleoside linkages present between the nucleobase units of the contiguous nucleotide sequence are phosphorothioate internucleoside linkages. 19. The pharmaceutical composition according to any one of embodiments 1-18, wherein said oligomer is conjugated with one or more non-nucleobase compounds. 20. The pharmaceutical composition according to any one of embodiments 1-19, wherein the contiguous nucleotide sequence is complementary to the corresponding sequence of at least two miRNA sequences such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA sequences. 21. The pharmaceutical composition according to any one of embodiments 1-20, wherein the contiguous nucleotide sequence consists or comprises of a sequence which is complementary to the sequence of at least two miRNA seed region sequences such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA seed region sequences. 22. The pharmaceutical composition according to any one of embodiments 20 or 21, wherein the contiguous nucleotide sequence is complementary to the corresponding region of both miR-221 and miR-222. 23. The pharmaceutical composition according to embodiment 22, wherein the contiguous nucleotide sequence consists or comprises of a sequence that is complementary to 5′GCUACAU3′. 24. The pharmaceutical composition according to any one of embodiments 1-23, wherein the oligomer is constituted as a prodrug. 25. The pharmaceutical composition according to any one of embodiments 1-24, wherein the contiguous nucleotide sequence is complementary to a corresponding region of has-miR-122. 26. The pharmaceutical composition according to embodiment 25, for use in the treatment of a medical disorder or disease selected from the group consisting of: hepatitis C virus infection and hypercholesterolemia and related disorders. 27. The pharmaceutical composition according to embodiment 25 or 26, wherein the composition further comprises a second independent active ingredient that is an inhibitor of the VLDL assembly pathway, such as an ApoB inhibitor, or an MTP inhibitor. 28. A kit comprising a pharmaceutical composition according to embodiment 25 or 26, and a second independent active ingredient that is an inhibitor of the VLDL assembly pathway, such as an ApoB inhibitor, or an MTP inhibitor. 29. A method for the treatment of a disease or medical disorder associated with the presence or overexpression of a microRNA, comprising the step of administering a the pharmaceutical composition) according to any one of embodiments 1-28 to a patient who is suffering from, or is likely to siffer from said disease or medical disorder. 30. An oligomer, as defined according to anyone of embodiments 1-25. 31. A conjugate comprising the oligomer according to embodiment 30, and at least one non-nucleobase compounds. 32. The use of an oligomer or a conjugate as defined in any one of embodiments 30-31, for the manufacture of a medicament for the treatment of a disease or medical disorder associated with the presence or over-expression of the microRNA. 33. A method for reducing the amount, or effective amount, of a miRNA in a cell, comprising administering an oligomer, a conjugate or a pharmaceutical composition, according to any one of the preceeding embodiments to the cell which is expressing said miRNA so as to reduce the amount, or effective amount of the miRNA in the cell. 34. A method for de-repression of a mRNA whose expression is repressed by a miRNA in a cell comprising administering an oligomer, a conjugate or a pharmaceutical composition, according to any one of the preceeding embodiments to the cell to the cell which expressed both said mRNA and said miRNA, in order to de-repress the expression of the mRNA. References: Details of the reference are provided in the priority documents.

EXAMPLES

LNA Monomer and oligonucleotide synthesis were performed using the methodology referred to in Examples 1 and 2 of WO2007/112754. The stability of LNA oligonucletides in human or rat plasma is performed using the methodology referred to in Example 4 of WO2007/112754. The treatment of in vitro cells with LNA anti-miR antisense oligonucleotide (targeting miR-122) is performed using the methodology referred to in Example 6 of WO2007/112754. The analysis of Oligonucleotide Inhibition of miR expression by microRNA specific quantitative PCR in both an in vitro and in vivo model is performed using the methodology referred to in Example 7 of WO2007/112754. The assessment of LNA antimir knock-down specificity using miRNA microarray expression profiling is performed using the methodology referred to in Example 8 of WO2007/112754. The detection of microRNAs by in situ hybridization is performed using the methodology referred to in Example 9 of WO2007/112754. The Isolation and analysis of mRNA expression (total RNA isolation and cDNA synthesis for mRNA analysis) in both an in vitro and in vivo model is performed using the methodology referred to in Example 10 of WO2007/112754. In vivo Experiments using Oligomers of the invention targeting microRNA-122. and subsequent analysis are performed using the methods disclosed in Examples 11-27 of WO2007/112754. The above mentioned examples of WO2007/112754 are hereby specifically incorporated by reference.

Example 1 Design of the LNA antimiR Oligonucleotides and Melting Temperatures

TABLE 2 Oligomers used in the examples and figures. The Compound # is an identifier used throughout the examples and figures - the SEQ ID NO which is used in the sequence listing is also provided. SEQ ID Compound NO Compound Sequence Comment 3204 1 TcAGtCTGaTaAgCT 3205 2 GATAAGCT 3206 3 TcAcAATtaGCAtTA 3207 4 TAGCATTA    4 5 CcAttGTcaCaCtCC 3208 6 CACACTCC 3209 7 TAAGCT 3210 8 ATAAGCT 3211 9 TGATAAGCT 3212 10 CTGATAAGCT 3213 11 GTCTGATAAGCT 3214 12 CAGTCTGATAAGCT 3215 13 TCTGATAA 3216 14 ATCAGTCT 3217 15 TCAACATC 3218 or 3230 16 G G TAA A CT Underline = mismatch 3219 17 CG TAA TGA Underline = mismatch 3220 18 TCAgtctgataaGCTa 5′ fluorescent label (FAM) 3221 19 AGCACTTT 3222 20 ATTTGCAC 3223 21 AgCagACaaTgTaGC 5′ fluorescent label (FAM) 3224 22 GtAgcCAgaTgTaGC 5′ fluorescent label (FAM) 3225 23 ATGTAGC 3226 24 ACaAcCTacTaCcTC 3227 25 ACTACCTC 3228 26 CaCtgTCagCaCtTT 3229 27 TgCatAGatTtGcAC 3231 28 GTAGACT 3232 29 TACCTC 3233 30 CTACCTC 3234 31 TNCTACCTC N = universal base. 3235 32 TNCTACCTC N = universal base. 3236 33 GCaAcCTacTaCcTC 3237 34 ACaAcCTccTaCcTC 3238 35 ACaAaCTacTaCcTC 3239 36 CTACCTC 3240 37 CTAACTC 3241 38 TTAGCATTA 3242 39 CGATTAGCATTA 3243 977 CACGATTAGCATTA 3244 978 GCATTA 3245 979 AGCATTA 3246 980 ATTAGCATTA Capital and lower case letters denote LNA and DNA, respectively. LNA cytosines are preferably methyl cytosine/5'methyl-cytosine* All internucleoside linkages are preferably phosphorothioate* All LNA may, for example, be beta-D-oxy LNA* *Used in the specific examples.

Example 2 In vitro Model: Cell Culture

The effect of LNA oligonucleotides on target nucleic acid expression (amount) can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. Target can be expressed endogenously or by transient or stable transfection of a nucleic acid encoding said nucleic acid.

The expression level of target nucleic acid can be routinely determined using, for example, Northern blot analysis (including microRNA northern), Quantitative PCR (including microRNA qPCR), Ribonuclease protection assays. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen.

Cells were cultured in the appropriate medium as described below and maintained at 37° C. at 95-98% humidity and 5% CO₂. Cells were routinely passaged 2-3 times weekly.

15PC3: The human prostate cancer cell line 15PC3 was kindly donated by Dr. F. Baas, Neurozintuigen Laboratory, AMC, The Netherlands and was cultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+Glutamax I+gentamicin.

PC3: The human prostate cancer cell line PC3 was purchased from ATCC and was cultured in F12 Coon's with glutamine (Gibco)+10% FBS+gentamicin.

518A2: The human melanoma cancer cell line 518A2 was kindly donated by Dr. B. Jansen, Section of experimental Oncology, Molecular Pharmacology, Department of Clinical Pharmacology, University of Vienna and was cultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+Glutamax I+gentamicin. HeLa: The cervical carcinoma cell line HeLa was cultured in MEM (Sigma) containing 10% fetal bovine serum gentamicin at 37° C., 95% humidity and 5% CO₂. MPC-11: The murine multiple myeloma cell line MPC-11 was purchased from ATCC and maintained in DMEM with 4 mM Glutamax+10% Horse Serum. DU-145: The human prostate cancer cell line DU-145 was purchased from ATCC and maintained in RPMI with Glutamax+10% FBS. RCC-4 +/− VHL: The human renal cancer cell line RCC4 stably transfected with plasmid expressing VHL or empty plasmid was purchased from ECACC and maintained according to manufacturers instructions. 786-0: The human renal cell carcinoma cell line 786-0 was purchased from ATCC and maintained according to manufacturers instructions HUVEC: The human umbilical vein endothelial cell line HUVEC was purchased from Camcrex and maintained in EGM-2 medium. K562: The human chronic myelogenous leukaemia cell line K562 was purchased from ECACC and maintained in RPMI with Glutamax+10% FBS. U87MG: The human glioblastoma cell line U87MG was purchased from ATCC and maintained according to the manufacturers instructions. B16: The murine melanoma cell line B16 was purchased from ATCC and maintained according to the manufacturers instructions. LNCap: The human prostate cancer cell line LNCap was purchased from ATCC and maintained in RPMI with Glutamax+10% FBS Huh-7: Human liver, epithelial like cultivated in Eagles MEM with 10% FBS, 2 mM Glutamax I, 1× non-essential amino acids, Gentamicin 25 μg/ml L428: (Deutsche Sammlung für Mikroorganismen (DSM, Braunschwieg, Germany)): Human B cell lymphoma maintained in RPMI 1640 supplemented with 10% FCS, L-glutamine and antibiotics. L1236: (Deutsche Sammlung für Mikroorganismen (DSM, Braunschwieg, Germany)): Human B cell lymphoma maintained in RPMI 1640 supplemented with 10% FCS, L-glutamine and antibiotics.

Example 3 Design of a LNA antimiR Library for all Human microRNA Sequences in miRBase microRNA Database

The miRBase version used was version 12, as reported in Griffiths-Jones, S., Grocock, R. J., van Dongen, S., Bateman, A., Enright, A. J. 2006. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res. 34: D140-4, and available via http://microrna.sanger.ac.uk/sequences/index.shtml.

Table 1 shows 7-, 8- and 9-mer nucleotide sequences comprising the seedmer sequence of micro RNA's according to the miRBase micro RNA database. The seedmer sequence comprises the reverse complement of the microRNA seed region. In some emboidments the oligomer of the invention has a contiguous nucleotide sequence selected from the 7-mer, 8-mer or 9-mer sequences. With respect to the 7-mer, 8-mer and 9-mer sequences, in some embodiments, all the internucleoside linkages are phosphorothioate. The 7-mer, 8-mer and 9-mer nucleotide sequences may consist of sequence of nucleotide analogues as described herein, such as LNA nucleotide analogues. LNA cytosines may be methyl-cytosine (5′ methyl-cytosine). In some embodiments, the LNA is beta-D-oxy-LNA.

Table 3 provides a list of microRNAs grouped into those which can be targeted by the same seedmer oligomers, such as the 7-, 8- or 9-mers provided herein (see table 1).

TABLE 3 hsa-let-7a* (SEQ ID NO: 97), hsa-let-7f-1* (SEQ ID NO: 107) hsa-let-7a (SEQ ID NO: 96), hsa-let-7b (SEQ ID NO: 98), hsa-let-7c (SEQ ID NO: 100), hsa-let-7d (SEQ ID NO: 102), hsa-let-7f (SEQ ID NO: 106), hsa-miR-98 (SEQ ID NO: 947), hsa-let-7g (SEQ ID NO: 109), hsa-let-7i (SEQ ID NO: 111) hsa-miR-1 (SEQ ID NO: 113), hsa-miR-206 (SEQ ID NO: 403) hsa-miR-103 (SEQ ID NO: 118), hsa-miR-107 (SEQ ID NO: 126) hsa-miR-10a (SEQ ID NO: 127), hsa-miR-10b (SEQ ID NO: 129) hsa-miR-125b (SEQ ID NO: 190), hsa-miR-125a-5p (SEQ ID NO: 189) hsa-miR-129* (SEQ ID NO: 229), hsa-miR-129-3p (SEQ ID NO: 230) hsa-miR-130a (SEQ ID NO: 251), hsa-miR-301a (SEQ ID NO: 473), hsa-miR-130b (SEQ ID NO: 253), hsa-miR-454 (SEQ ID NO: 608), hsa-miR-301b (SEQ ID NO: 474) hsa-miR-133a (SEQ ID NO: 261), hsa-miR-133b (SEQ ID NO: 262) hsa-miR-135a (SEQ ID NO: 264), hsa-miR-135b (SEQ ID NO: 266) hsa-miR-141 (SEQ ID NO: 278), hsa-miR-200a (SEQ ID NO: 392) hsa-miR-146a (SEQ ID NO: 290), hsa-miR-146b-5p (SEQ ID NO: 293) hsa-miR-152 (SEQ ID NO: 308), hsa-miR-148b (SEQ ID NO: 300) hsa-miR-154* (SEQ ID NO: 314), hsa-miR-487a (SEQ ID NO: 619) hsa-miR-15a (SEQ ID NO: 317), hsa-miR-16 (SEQ ID NO: 321), hsa-miR-15b (SEQ ID NO: 319), hsa-miR-195 (SEQ ID NO: 377), hsa-miR-497 (SEQ ID NO: 634) hsa-miR-17 (SEQ ID NO: 324), hsa-miR-20a (SEQ ID NO: 406), hsa-miR-93 (SEQ ID NO: 930), hsa-miR-106a (SEQ ID NO: 122), hsa-miR-106b (SEQ ID NO: 124), hsa-miR-20b (SEQ ID NO: 408), hsa-miR-526b* (SEQ ID NO: 708) hsa-miR-181a (SEQ ID NO: 326), hsa-miR-181c (SEQ ID NO: 330) hsa-miR-181b (SEQ ID NO: 329), hsa-miR-181d (SEQ ID NO: 332) hsa-miR-18a (SEQ ID NO: 349), hsa-miR-18b (SEQ ID NO: 351) hsa-miR-190 (SEQ ID NO: 353), hsa-miR-190b (SEQ ID NO: 357) hsa-miR-192 (SEQ ID NO: 369), hsa-miR-215 (SEQ ID NO: 417) hsa-miR-196a (SEQ ID NO: 379), hsa-miR-196b (SEQ ID NO: 381) hsa-miR-199a-3p, hsa-miR-199b-3p (SEQ ID NO: 385) hsa-miR-199a-5p (SEQ ID NO: 384), hsa-miR-199b-5p (SEQ ID NO: 386) hsa-miR-19a* (SEQ ID NO: 388), hsa-miR-19b-1* (SEQ ID NO: 390), hsa-miR-19b-2* (SEQ ID NO: 391) hsa-miR-19a (SEQ ID NO: 387), hsa-miR-19b (SEQ ID NO: 389) hsa-miR-200b (SEQ ID NO: 394), hsa-miR-200c (SEQ ID NO: 396) hsa-miR-204 (SEQ ID NO: 401), hsa-miR-211 (SEQ ID NO: 413) hsa-miR-208a (SEQ ID NO: 404), hsa-miR-208b (SEQ ID NO: 405) hsa-miR-212 (SEQ ID NO: 414), hsa-miR-132 (SEQ ID NO: 255) hsa-miR-23a* (SEQ ID NO: 440), hsa-miR-23b* (SEQ ID NO: 442) hsa-miR-23a (SEQ ID NO: 439), hsa-miR-23b (SEQ ID NO: 441), hsa-miR-130a* (SEQ ID NO: 252) hsa-miR-24-1* (SEQ ID NO: 444), hsa-miR-24-2* (SEQ ID NO: 445) hsa-miR-25 (SEQ ID NO: 446), hsa-miR-92a (SEQ ID NO: 925), hsa-miR-367 (SEQ ID NO: 547), hsa-miR-92b (SEQ ID NO: 928) hsa-miR-26a (SEQ ID NO: 448), hsa-miR-26b (SEQ ID NO: 451) hsa-miR-26a-1* (SEQ ID NO: 449), hsa-miR-26a-2* (SEQ ID NO: 450) hsa-miR-27a (SEQ ID NO: 453), hsa-miR-27b (SEQ ID NO: 455) hsa-miR-29a (SEQ ID NO: 465), hsa-miR-29b (SEQ ID NO: 467), hsa-miR-29c (SEQ ID NO: 470) hsa-miR-302a (SEQ ID NO: 475), hsa-miR-302b (SEQ ID NO: 477), hsa-miR-302c (SEQ ID NO: 479), hsa-miR-302d (SEQ ID NO: 481), hsa-miR-373 (SEQ ID NO: 555), hsa-miR-520e (SEQ ID NO: 696), hsa-miR-520a-3p (SEQ ID NO: 690), hsa-miR-520b (SEQ ID NO: 692), hsa-miR-520c-3p (SEQ ID NO: 693), hsa-miR-520d-3p (SEQ ID NO: 694) hsa-miR-302b* (SEQ ID NO: 478), hsa-miR-302d* (SEQ ID NO: 482) hsa-miR-30a* (SEQ ID NO: 486), hsa-miR-30d* (SEQ ID NO: 493), hsa-miR-30e* (SEQ ID NO: 495) hsa-miR-30a (SEQ ID NO: 485), hsa-miR-30c (SEQ ID NO: 489), hsa-miR-30d (SEQ ID NO: 492), hsa-miR-30b (SEQ ID NO: 487), hsa-miR-30e (SEQ ID NO: 494) hsa-miR-330-5p (SEQ ID NO: 513), hsa-miR-326 (SEQ ID NO: 509) hsa-miR-34a (SEQ ID NO: 534), hsa-miR-34c-5p (SEQ ID NO: 539), hsa-miR-449a (SEQ ID NO: 599), hsa-miR-449b (SEQ ID NO: 600) hsa-miR-362-3p (SEQ ID NO: 542), hsa-miR-329 (SEQ ID NO: 511) hsa-miR-374a (SEQ ID NO: 557), hsa-miR-374b (SEQ ID NO: 559) hsa-miR-376a (SEQ ID NO: 562), hsa-miR-376b (SEQ ID NO: 564) hsa-miR-378 (SEQ ID NO: 568), hsa-miR-422a (SEQ ID NO: 585) hsa-miR-379* (SEQ ID NO: 571), hsa-miR-411* (SEQ ID NO: 582) hsa-miR-381 (SEQ ID NO: 574), hsa-miR-300 (SEQ ID NO: 472) hsa-miR-509-5p (SEQ ID NO: 655), hsa-miR-509-3-5p (SEQ ID NO: 653) hsa-miR-515-5p (SEQ ID NO: 666), hsa-miR-519e* (SEQ ID NO: 689) hsa-miR-516b*, hsa-miR-516a-3p (SEQ ID NO: 667) hsa-miR-517a (SEQ ID NO: 671), hsa-miR-517c (SEQ ID NO: 673) hsa-miR-518a-5p (SEQ ID NO: 674), hsa-miR-527 (SEQ ID NO: 709) hsa-miR-518f (SEQ ID NO: 681), hsa-miR-518b (SEQ ID NO: 675), hsa-miR-518c (SEQ ID NO: 676), hsa-miR-518a-3p (SEQ ID NO: 674), hsa-miR-518d-3p (SEQ ID NO: 678) hsa-miR-519c-3p (SEQ ID NO: 686), hsa-miR-519b-3p (SEQ ID NO: 685), hsa-miR-519a (SEQ ID NO: 683) hsa-miR-519c-5p, hsa-miR-519b-5p, hsa-miR-523*, hsa-miR-518f* (SEQ ID NO: 682), hsa-miR-526a, hsa-miR-520c-5p, hsa-miR-518e*, hsa-miR-518d-5p (SEQ ID NO: 679),hsa-miR-522*, hsa-miR-519a* (SEQ ID NO: 684) hsa-miR-519e (SEQ ID NO: 688), hsa-miR-33b* (SEQ ID NO: 527) hsa-miR-520a-5p (SEQ ID NO: 691), hsa-miR-525-5p (SEQ ID NO: 706) hsa-miR-520g (SEQ ID NO: 698), hsa-miR-520h (SEQ ID NO: 699) hsa-miR-524-5p (SEQ ID NO: 704), hsa-miR-520d-5p (SEQ ID NO: 695) hsa-miR-525-3p (SEQ ID NO: 705), hsa-miR-524-3p (SEQ ID NO: 703) hsa-miR-548b-5p (SEQ ID NO: 724), hsa-miR-548a-5p (SEQ ID NO: 722), hsa-miR-548c-5p (SEQ ID NO: 726), hsa-miR-548d-5p (SEQ ID NO: 728) hsa-miR-7-1* (SEQ ID NO: 880), hsa-miR-7-2* (SEQ ID NO: 881) hsa-miR-99a (SEQ ID NO: 948), hsa-miR-100 (SEQ ID NO: 114), hsa-miR-99b (SEQ ID NO: 950) We have constructed an 8-mer LNA-antimiR against miR-21, miR-155 and miR-122 (designated here as micromiR) that is fully LNA modified and phosphorothiolated (see FIG. 1 and Table 6). Our results from repeated experiments in MCF-7, HeLa, Raw and Huh-7 cells using a luciferase sensor plasmid for miR-21, miR-155 and miR-122 demonstrate that the fully LNA-modified short LNA-antimiRs are highly potent in antagonizing microRNAs.

TABLE 4 LNA_antimiR & MicromiR sequences and predicted T_(m)s SEQ micro T_(m) Compound ID NO RNA Sequence (° C.) 3204 1 miR-21 T c A G t C T G a T a A g C T 73 3205 2 G A T A A G C T 33 3206 3 miR-155 T c A c A A T t a G C A t T A 63 3207 4 T A G C A T T A 45 4 5 miR-122 C c A t t G T c a C a C t C C 73 3208 6 C A C A C T C C 62 Capital letters are LNA units, such as beta-D-oxy LNA. Lower case letters are DNA units. Internucleoside linkages are preferably phosphorothioate. LNA cytosines are all preferably methylated/5-methyl cytosine. The melting temperatures can be assessed towards the mature microRNA sequence, using a synthetic microRNA oligonucleotide (typically consisting of RNA nucleotides with a phosphodiester backbone). Typically measured T_(m)s are higher than predicted T_(m)s when using LNA oligomers against the RNA target.

Example 4 Assessment of miR-21 Antagonism by Compound 3205 (SEQ ID NO: 2) LNA-antimiR in MCF-7 Cells Using a Luciferase Sensor Assay

In order to assess the efficiency of a fully LNA-modified 8-mer LNA-antimiR (Compound 3205, SEQ ID NO: 2) oligonucleotide in targeting and antagonizing miR-21, luciferase sensor constructs were made containing a perfect match target site for the mature miR-21 and as control, a target site with two mutations in the seed (FIG. 6). In order to monitor microRNA-21 inhibition, the breast carcinoma cell line MCF-7 was transfected with the different luciferase constructs together with the miR-21 antagonist Compound 3205 (SEQ ID NO: 2) at varying concentrations in comparison with a 15-mer LNA-antimiR Compound 3204 (SEQ ID NO: 1) against miR-21. After 24 hours, luciferase activity was measured.

Results:

As seen in FIG. 2, the new fully LNA-modified 8-mer LNA-antimiR (Compound 3205 (SEQ ID NO: 2) shows two-fold higher potency compared to Compound 3204 (SEQ ID NO: 1), as shown by de-repression of the luciferase activity. By contrast, the control miR-21 sensor construct with two mismatches in the miR-21 seed did not show any de-repression of the firefly luciferase activity, thereby demonstrating the specificity of the perfect match miR-21 sensor in monitoring miR-21 activity in cells. The de-repression of luciferase activity by the 8-mer LNA-antimiR is clearly dose-dependent, which is not seen with Compound 3204 (SEQ ID NO: 1). Moreover, the new 8-mer is also much more potent at lower doses than Compound 3204 (SEQ ID NO: 1).

To conclude, the 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) shows significantly improved potency in inhibition of miR-21 in vitro compared to the 15-mer LNA-antimiR Compound 3204 (SEQ ID NO: 1) targeting miR-21.

Materials and Methods:

Cell Line:

The breast carcinoma cell line MCF-7 was purchased from ATCC (#HTB-22™). MCF-7 cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

400.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50-70% confluency the next day. On the day of transfection, MCF-7 cells were transfected with 0.8 ug miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector (SDS Promega) together with 1 μl Lipofectamine-2000 (Invitrogen) according to manufacturer's instructions. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and harvested with cell scraper, after which cells were centrifugated for 5 min at 10.000 rpm. The supernatant was discarded and 50 μl 1× Passive Lysis Buffer (Promega) was added to the cell pellet, after which cells were put on ice for 30 min. The lysed cells were spinned at 10.000 rpm for 30 min after which 20 μl were transferred to a 96 well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).

Example 5 Assessment of miR-21 Antagonism by Compound 3205 (SEQ ID NO: 2) LNA-antimiR in HeLa Cells Using a Luciferase Sensor Assay

To further assess the efficiency of the fully LNA-modified 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) in targeting miR-21, the cervix carcinoma cell line HeLa was also transfected with the previously described miR-21 luciferase sensor constructs alongside Compound 3205 (SEQ ID NO: 2) at varying concentrations as described in the above section (FIG. 3).

Results:

Compound 3205 (SEQ ID NO: 2) shows complete de-repression of the miR-21 luciferase sensor construct in HeLa cells already at 5 nM compared to Compound 3204 (SEQ ID NO: 1), which did not show complete de-repression until the highest dose (50 nM). In addition, antagonism of miR-21 by the 8-mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR is dose-dependent. To demonstrate the specificity of the miR-21 luciferase sensor assay, a mismatched miR-21 target site (2 mismatches in seed) was also transfected into HeLa cells, but did not show any de-repression of the firefly luciferase activity.

To conclude, the fully LNA-modified Compound 3205 (SEQ ID NO: 2) shows significantly improved potency in inhibition of miR-21 in vitro, in both MCF-7 and HeLa cells compared to the 15-mer LNA-antimiR Compound 3204 (SEQ ID NO: 1).

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24 well plate the day before transfection in order to receive 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 0.2 ug miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector together with 0.7 μl Lipofectamine-2000 (Invitrogen) according to manufacturer's instructions. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24 well plates was put on an orbital shaker for 30 min. The cells were collected and transferred to an eppendorf tube and spinned at 10.000 rpm for 30 min after which 10 μl were transferred to a 96 well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).

Example 6 Assessment of miR-155 Antagonism by Compound 3207 (SEQ ID NO: 4) LNA-antimiR in Mouse RAW Cells Using a Luciferase Sensor Assay

To ask whether a fully LNA-modified 8-mer LNA-antimiR can effectively antagonize miR-155, a perfect match target site for miR-155 was cloned into the same luciferase vector (psiCHECK2) and transfected into the mouse leukaemic monocyte macrophage RAW cell line. Because the endogenous levels of miR-155 are low in the RAW cell line, the cells were treated with 100 ng/ml LPS for 24 hours in order to induce miR-155 accumulation.

Results:

Luciferase measurements showed that the fully LNA-modified 8-mer LNA-antimiR Compound 3207 (SEQ ID NO: 4) targeting miR-155 was similarly effective in antagonizing miR-155 compared to the 15-mer LNA-antimiR Compound 3206 (SEQ ID NO: 3) (FIG. 4). Both LNA-antimirs showed a >50% de-repression of the miR-155 luciferase sensor at 0.25 nM concentration and inhibited miR-155 in a dose-dependent manner.

Conclusion:

These data further support the results from antagonizing miR-21, as shown in examples 1 and 2, demonstrating that a fully thiolated 8-mer LNA-antimiR is highly potent in microRNA targeting.

Materials and Methods:

Cell Line:

The mouse leukaemic monocyte macrophage RAW 264.7 was purchased from ATCC (TIB-71). RAW cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

500.000 cells were seeded per well in a 6 well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, MCF-7 cells were transfected with 0.3 ug miR-155 or empty psiCHECK2 vector together with 10 μl Lipofectamine-2000 (Invitrogen) according to manufacturer's instructions. In order to induce miR-155 accumulation, LPS (100 ng/ml) was added to the RAW cells after the 4 hour incubation with the transfection complexes. After another 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and harvested with cell scraper, after which cells were centrifugated for 5 min at 2.500 rpm. The supernatant were discarded and 50 μl 1+ Passive Lysis Buffer (Promega) was added to the cell pellet, after which cells were put on ice for 30 min. The lysed cells were spinned at 10.000 rpm for 30 min after which 20 μl were transferred to a 96 well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).

Example 7 Assessment of miR-122 Antagonism by Compound 3208 (SEQ ID NO: 6) LNA-antimiR in HuH-7 Cells Using a Luciferase Sensor Assay

The potency of the fully modified 8-mer LNA-antimiR Compound 3208 (SEQ ID NO: 6) against miR-122 was assessed in the human hepatoma cell line HuH-7. The HuH-7 cells were transfected with luciferase sensor construct containing a perfect match miR-122 target site. After 24 hours luciferase measurements were performed (FIG. 5).

Results:

The fully LNA-modified 8-mer LNA-antimiR Compound 3208 (SEQ ID NO: 6) is more potent than the 15-mer LNA-antimiR Compound 4 (SEQ ID NO:5) at low concentration, as shown by de-repression of the miR-122 luciferase sensor. Both LNA-antimiRs inhibit miR-122 in a dose-dependent manner (FIG. 5).

Conclusion:

The fully LNA-modified 8-mer LNA-antimiR Compound 3208 (SEQ ID NO: 6) targeting miR-122 shows improved potency in inhibition of miR-122 in vitro.

Materials and Methods:

Cell Line:

The human hepatoma cell line HuH-7 was a kind gift from R. Bartenschlager, Heidelberg. Huh-7 cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

8.000 cells were seeded per well in a 96 well plate the day before transfection in order to receive 50-70% confluency the next day. On the day of transfection, HuH-7 cells were transfected with 57 ng miR-122 or empty psiCHECK2 vector together with 1 μl Lipofectamine-2000 (Invitrogen). After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

50 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 96 well plate was put on an orbital shaker for 30 min. To each well the Dual-luciferase Reporter assay system (Promega) was added and luciferase measurements were performed according to manufacturer's instructions (Promega).

Example 8 Assessment of miR-21 Antagonism by Comparing an 8-Mer (Compound 3205, SEQ ID NO: 2) versus a 15-mer (Compound 3204, SEQ ID NO: 1) LNA-antimiR in Human Prostate Carcinoma Cells (PC3)

We have previously shown (patent application 1051), that an 8-mer LNA-antimiR that is fully LNA-modified and phosphorothiolated is able to completely de-repress the miR-21 luciferase reporter levels in the human cervix carcinoma cell line HeLa and partly de-repress the miR-21 luciferase reporter levels in the human breast carcinoma cell line MCF-7. We next extended this screening approach to the human prostate cancer cell line PC3. To assess the efficiency of the different LNA-antimiR oligonucleotides against miR-21, luciferase reporter constructs were generated in which a perfect match target site for the mature miR-21 and a target site with two mismatches in the seed were cloned in the 3′UTR of Renilla luciferase gene (FIG. 7). In order to monitor miR-21 inhibition, PC3 cells were transfected with the different luciferase constructs together with the miR-21 antagonist Compound 3205 (SEQ ID NO: 2) (8-mer) and for comparison with the 15-mer LNA-antimiR perfect match Compound 3204 (SEQ ID NO: 1) at varying concentrations. After 24 hours, luciferase activity was measured.

Results:

The luciferase reporter experiments showed a dose-dependent de-repression of the luciferase miR-21 reporter activity with the 15-mer LNA-antimiR against miR-21 (Compound 3204, SEQ ID NO: 1). However, complete de-repression of the luciferase reporter was not obtained even at the highest concentrations (FIG. 7). In contrast, the cells that were transfected with the 8-mer fully LNA substituted LNA-antimiR showed complete de-repression already at 1 nM, indicating significantly improved potency compared to the 15-mer LNA-antimiR. The luciferase control reporter harboring a mismatch target site for miR-21 was not affected by either LNA-antimiR, demonstrating high specificity of both LNA-antimiRs.

Conclusion:

The micromer is far more potent than the 15-mer LNA-antimiR in targeting miR-21 and has so far shown to be most potent in prostate carcinoma cells.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC (#90112714). PC3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

100.000 cells were seeded per well in a 12-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, PC3 cells were transfected with 0.3 μg miR-21 or empty psiCHECK2 vector together with 1.2 μl Lipofectamine-2000 (Invitrogen) according to manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 250 μl 1× Passive Lysis Buffer (Promega) was added to the wells. The plates were placed on a shaker for 30 min., after which the cell lysates were transferred to eppendorf tubes. The cell lysate was centrifugated for 10 min at 2.500 rpm after which 20 μl were transferred to a 96 well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).

Example 9 Specificity Assessment of miR-21 Antagonism by an 8-Mer LNA-antimiR

To investigate the specificity of our short LNA-antimiR targeting miR-21, we designed an 8-mer mismatch control LNA-antimiR (Compound 3218, SEQ ID NO: 16) containing 2 mismatches in the seed recognition sequence (see FIG. 8). The luciferase reporter constructs described in example 1 were transfected into the human cervix carcinoma cell line HeLa together with the LNA mismatch control oligo Compound 3218 (SEQ ID NO: 16) and its efficacy was compared with the 8-mer LNA-antimiR (Compound 3205, SEQ ID NO: 2) targeting miR-21. After 24 hours, luciferase activity was measured.

Results:

As shown in FIG. 8, transfection of the fully LNA-modified 8-mer LNA-antimiR in HeLa cells resulted in complete de-repression of the luciferase miR-21 reporter already at 5 nM. In contrast, when the cells were transfected with the 8-mer LNA mismatch control oligo, combined with the results obtained with the control miR-21 luciferase reporter having two mismatches in the miR-21 seed, these data demonstrate high specificity of the fully LNA-substituted 8-mer LNA-antimiR in targeting miR-21 in Hela cells.

Analysis of the miRBase microRNA sequence database showed that the miR-21 recognition sequence, of the LNA-antimiR Compound 3205 (SEQ ID NO: 2) is unique for microRNA-21. However, when decreasing the micromer length to 7 nt, it is not specific for only miR-21, since ath-miR-844, mmu-miR-590-3p and has-miR-590-3p are also targeted.

Conclusion:

Exchanging two nucleotide positions within the 8-mer LNA-antimiR with two mismatching nucleotides completely abolished the antagonizing activity of the LNA-antimiR for miR-21.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24-well plate the day before transfection in order to receive 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 0.2 ug miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector together with 0.7 μl Lipofectamine-2000 (Invitrogen) according to manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24-well plates were put on an orbital shaker for 30 min. The cells were collected and transferred to an eppendorf tube and spinned at 10.000 rpm for 30 min after which 10 μl were transferred to a 96-well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).

Example 10 Assessment of the Shortest Possible Length of a Fully LNA-Modified LNA-antimiR that Mediates Effective Antagonism of miR-21

To further investigate the LNA-antimiR length requirements, we designed a 7-mer and a 6-mer LNA-antimiR targeting miR-21, both fully LNA-modified and phosphorothiolated oligonucleotides. The miR-21 luciferase reporter constructs were transfected into HeLa cells along with the LNA-antimiRs at varying concentrations. Luciferase measurements were performed after 24 hours.

Results:

As seen in FIG. 9, the 7-mer LNA-antimiR mediates de-repression of the miR-21 luciferase reporter plasmid, but at lower potency compared to the 8-mer LNA-antimiR (Compound 3205, SEQ ID NO: 2). Nevertheless, a dose-dependent trend can still be observed. By contrast, the 6-mer LNA-antimiR did not show any inhibitory activity.

Conclusion:

To conclude, the shortest possible length of an LNA-antimiR which is able to mediate miR-21 inhibition is 7 nucleotides. However, the 7-mer LNA-antimiR is less potent compared to the 8-mer LNA-antimiR for miR-21.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24 well plate the day before transfection in order to receive 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 0.2 ug miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector together with 0.7 μl Lipofectamine-2000 (Invitrogen) according to manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24-well plates was put on an orbital shaker for 30 min. The cells were collected and transferred to an eppendorf tube and spinned at 10.000 rpm for 30 min after which 10 μl were transferred to a 96-well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).

Example 11 Length Assessment of Fully LNA-Substituted LNA-antimiRs Antagonizing miR-21

Next, we investigated the effect of increasing the length from a 9-mer to a 14-mer fully LNA substituted LNA-antimiRs on antagonizing miR-21 in HeLa cells. The resulting LNA-antimiRs were transfected into HeLa cells together with the miR-21 luciferase reporter constructs (FIG. 10). Luciferase measurements were performed after 24 hours.

Results:

The 9-mer LNA-antimiR Compound 3211 (SEQ ID NO: 9) (9-mer) showed dose-dependent de-repression of the miR-21 luciferase reporter which did not reach complete de-repression, as demonstrated for the 7-mer LNA-antimiR (Compound 3210, SEQ ID NO: 8). Increasing the length to 10-mer to 14-mer (Compound 3212, (SEQ ID NO: 10); Compound 3213 (SEQ ID NO: 11); and Compound 3214 (SEQ ID NO: 12)) decreased the potency as shown by less efficient de-repression of the miR-21 reporter.

Conclusion:

As shown in FIG. 10, the longest fully LNA-modified and phosphorothiolated LNA-antimiR which is still able to mediate miR-21 inhibition is a 9-mer LNA-antimiR Compound 3211 (SEQ ID NO: 9). However, it is clearly less efficient than the 7-mer and 8-mer LNA-antimiRs.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24-well plate the day before transfection in order to achieve 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 0.2 ug miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 control vector without target site together with 0.7 μl Lipofectamine-2000 (Invitrogen) according to manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24-well plates were put on an orbital shaker for 30 min. The cells were collected and transferred to an eppendorf tube and spinned at 10.000 rpm for 30 min after which 10 μl were transferred to a 96-well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).

Example 12 Determination of the Most Optimal Position for an 8-Mer LNA-antimiR within the miR Target Recognition Sequence

Our experiments have shown that the most potent fully LNA-modified phosphorothiolated LNA-antimiR is 8 nucleotides in length. To assess the most optimal position for an 8-mer LNA-antimiR within the miR target recognition sequence, we designed four different fully LNA-modified 8-mer LNA-antimiRs tiled across the mature miR-21 sequence as shown in FIG. 11. The different LNA-antimiRs were co-transfected together with the miR-21 luciferase reporter constructs into HeLa cells. Luciferase measurements were performed after 24 hours.

Results:

The only LNA-antimiR that mediated efficient silencing of miR-21 as measured by the luciferase reporter was Compound 3205 (SEQ ID NO: 2), which targets the seed region of miR-21. Neither Compound 3215 (SEQ ID NO: 13) which was designed to cover the 3′ end of the seed (50% seed targeting) did not show any effect, nor did the other two LNA-antimiRs Compound 3216 (SEQ ID NO: 14) or Compound 3217 (SEQ ID NO: 15), which were positioned to target the central region and the 3′end of the mature miR-21, respectively.

Conclusion:

The only 8-mer LNA-antimiR mediating potent silencing of miR-21 is the one targeting the seed of the miR-21.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24-well plate the day before transfection in order to achieve 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 0.2 ug miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector together with 0.7 μl Lipofectamine-2000 (Invitrogen) according to the manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24-well plates was put on an orbital shaker for 30 min. The cells were collected and transferred to an eppendorf tube and spinned at 10.000 rpm for 30 min after which 10 μl were transferred to a 96 well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).

Example 13 Validation of interaction of the miR-21 target site in the Pdcd-4-3′-UTR and miR-21 Using the 8-Mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR

The tumour suppressor protein Pdcd4 inhibits TPA-induced neoplastic transformation, tumour promotion and progression. Pdcd4 has also been shown to be upregulated in apoptosis in response to different inducers. Furthermore, downregulation of Pdcd4 in lung and colorectal cancer has also been associated with a poor patient prognosis. Recently, Asangani et al and Frankel et al showed that the Pdcd-4-3′-UTR contains a conserved target site for miR-21, and transfecting cells with an antimiR-21, resulted in an increase in Pdcd4 protein. We therefore constructed a luciferase reporter plasmid, harboring 313 nt of the 3′UTR region of Pdcd4 encompassing the aforementioned miR-21 target site, which was co-transfected together with different LNA-antimiRs into HeLa cells. The different LNA-antimiRs were; Compound 3205 (SEQ ID NO:2) (8-mer, perfect match) or Compound 3218 (SEQ ID NO: 16) (8-mer, mismatch). Luciferase measurements were performed after 24 hours.

Results:

As shown in FIG. 12, in cells transfected with the Pdcd4 3′UTR luciferase reporter and Compound 3205 (SEQ ID NO: 2), an increase in luciferase activity was observed, indicating interaction between the Pdcd4 3″UTR and miR-21. However, transfecting the cells with the mismatch compound, Compound 3218 (SEQ ID NO: 16), no change in luciferase activity was observed, which was expected since the compound does not antagonize miR-21. When comparing the 8-mer LNA-antimiR against two longer designed LNA-antimiRs, the short fully LNA-modified and phosphorothiolated LNA-antimiR was significantly more potent, confirming previous luciferase assay data.

Conclusion:

These data conclude that Compound 3205 (SEQ ID NO: 2), which antagonizes miR-21, can regulate the interaction between Pdcd4 3′UTR and miR-21.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24-well plate the day before transfection in order to achieve 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 0.2 ug Pdcd-4-3′UTR/psiCHECK2 or empty psiCHECK2 vector together with 0.7 μl Lipofectamine-2000 (Invitrogen) according to the manufacturer's instructions. Varying concentrations of the LNA-antimiR oligonucleotides were also transfected. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24-well plates was put on an orbital shaker for 30 min. The cells were collected and transferred to an eppendorf tube and spinned at 10.000 rpm for 30 min after which 10 μl were transferred to a 96 well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).

Example 14 Comparison of an 8-Mer LNA-antimiR (Compound 3207, SEQ ID NO: 4) with a 15-Mer LNA-antimiR (Compound 3206, SEQ ID NO: 3) in Antagonizing miR-155 in Mouse RAW Cells

To ask whether our approach of using short LNA-antimiRs could be adapted to targeting other miRNAs we designed a fully LNA-modified 8-mer LNA-antimiR against microRNA-155. A perfect match target site for miR-155 was cloned into the 3′UTR of the luciferase gene in the reporter plasmid psiCHECK2 and transfected into the mouse RAW macrophage cell line together with an 8-mer or a 15-mer LNA-antimiR. Because the endogenous levels of miR-155 are low in the RAW cell line, the cells were treated with 100 ng/ml LPS for 24 hours in order to induce miR-155 accumulation. After 24 hours, luciferase analysis was performed.

Results:

Luciferase measurements showed that the fully LNA-modified 8-mer LNA-antimiR Compound 3207 (SEQ ID NO: 4) targeting miR-155 was similarly effective in antagonizing miR-155 compared to the 15-mer LNA-antimiR Compound 3206 (SEQ ID NO: 3) (FIG. 13). Both LNA-antimiRs showed a >50% de-repression of the miR-155 luciferase sensor at 0.25 nM concentration and inhibited miR-155 in a dose-dependent manner.

Analysis of the miRBase microRNA sequence database showed that the miR-155 recognition sequence, of the LNA-antimiR Compound 3207 (SEQ ID NO: 4) is unique for microRNA-155. However, when decreasing the LNA-antimiR length to 7 nt, it is not specific for only miR-155, mdv1-miR-M4 and kshv-miR-K12-11 (SEQ ID NO: 963) is also targeted.

Conclusion:

A fully LNA-modified and phosphorothiolated 8-mer LNA-antimiR is equally potent compared with a 15-mer LNA-antimiR of a mixed LNA/DNA design in antagonizing miR-155. Thus, our approach of using short LNA-antimiRs can be readily adapted to targeting of other miRNAs

Materials and Methods:

Cell Line:

The mouse macrophage RAW 264.7 cell line was purchased from ATCC (TIB-71). RAW cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

500.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, RAW 264.7 cells were transfected with 0.3 ug miR-155 perfect match/psiCHECK2 or empty psiCHECK2 vector together with 10 μl Lipofectamine-2000 (Invitrogen) according to the manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. In order to induce miR-155 accumulation, LPS (100 ng/ml) was added to the RAW cells after the 4 hour incubation with the transfection complexes. After another 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and harvested with cell scraper, after which cells were spinned for 5 min at 2.500 rpm. The supernatant was discarded and 50 μl 1× Passive Lysis Buffer (Promega) was added to the cell pellet, after which cells were put on ice for 30 min. The lysed cells were spinned at 10.000 rpm for 30 min after which 20 μl were transferred to a 96-well plate and luciferase measurements were performed according to the manufacturer's instructions (Promega).

Example 15 Assessment of c/EBP_(β) Protein Levels as a Functional Readout for miR-155 Antagonism by Short LNA-antimiR (Compound 3207, SEQ ID NO: 4)

As a functional readout for miR-155 antagonism by short LNA-antimiR (Compound 3207, SEQ ID NO: 4) we determined the protein levels of a novel miR-155 target, c/EBP_(β). The mouse macrophage RAW cell line was transfected together with either an 8-mer (Compound 3207, SEQ ID NO: 4) or a 15-mer (Compound 3206, SEQ ID NO: 3) LNA-antimiR in the absence or presence of pre-miR-155. As mismatch controls for the 15-mer, Compound 4 (SEQ ID NO: 5) was used, which targets miR-122 and for the 8-mer Compound 3205 (SEQ ID NO: 2) was used, which targets miR-21. These two control miRNAs do not regulate c/EBP_(β) expression levels. LPS was used to induce miR-155 accumulation and cells were harvested after 16 hours with LPS. c/EBP_(β) has three isoforms; LIP, LAP and LAP* that were detected by Western blot analysis and the same membranes were re-probed with beta-tubulin as loading control.

Results:

Ratios were calculated for c/EBP_(β) LIP and beta-tubulin as indicated in FIG. 14. RAW cells that were transfected with the 15-mer LNA-antimiR and no pre-miR-155 all showed equal c/EBP_(β) LIP/beta-tubulin ratios, due to inhibition of miR-155 increases the c/EBP_(β) LIP levels (FIG. 14, left panel). By comparison, transfection of pre-miR-155 in RAW cells resulted in decreased c/EBP_(β) LIP levels as expected, if c/EBP_(β) was a miR-155 target, as shown in lanes with protein extracts from RAW cells treated with no LNA or a mismatch. However, protein extracts from RAW cells transfected with LNA-antimiR against miR-155, showed an increase of c/EBP_(β) LIP levels. The same experiments were also carried out with the 8-mer LNA-antimiR-155 (Compound 3207, SEQ ID NO: 4) and as shown in FIG. 14 (right panel) comparable results to those with the 15-mer LNA-antimiR Compound 3206 (SEQ ID NO: 3) were obtained.

Conclusion:

Antagonism of miR-155 using either an 8-mer or a 15-mer LNA-antimiR leads to de-repression of the direct target c/EBP_(β).

Materials and Methods:

Cell Line:

The mouse macrophage RAW 264.7 cell line was purchased from ATCC (TIB-71). RAW cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

500.000 cells were seeded per well in a 6-well plate the day before transfection in order to achieve 50% confluency the next day. On the day of transfection, RAW 264.7 cells were transfected with 5 nmol pre-miR-155 (Ambion) and/or 5 nM LNA-antimiR together with 10 μl Lipofectamine-2000 (Invitrogen) according to the manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. In order to induce miR-155 accumulation, LPS (100 ng/ml) was added to the RAW cells after the 4 hour incubation with the transfection complexes. After 16 hours, cells were harvested for protein extraction and western blot analysis.

Western Blot:

Cells were washed with PBS, trypsinated, transferred to eppendorf tubes and 250 μl lysis buffer (1×RIPA) was added. The cell lysate was placed on ice for 20 min and spinned at 10.000 rpm for 10 minutes. The protein concentration was measured with Coomassie Plus according to the manufacturer's instructions and 80 ug was loaded onto a 4-12% BIS-TRIS gel. The membrane was incubated overnight at 4° C. with the primary monoclonal mouse antibody C/EBP_(β) (Santa Cruz) with a 1:100 concentration. Immunoreactive bands were visualized with ECL Plus (Amersham).

Example 16 Antagonism of miR-106b by a fully LNA-modified 8-mer (Compound 3221, SEQ ID NO: 19) LNA-antimiR

To confirm that our approach of using short LNA-antimiRs could be adapted to targeting of other miRNAs we designed a fully LNA-modified 8-mer LNA-antimiR against microRNA-106b. A perfect match target site for miR-106b was cloned into the 3′UTR of the luciferase gene in the vector (psiCHECK2) and transfected into the human cervix carcinoma HeLa cell line together with a short LNA-antimiR (Compound 3221, SEQ ID NO: 19) or with a 15-mer LNA-antimiR (Compound 3228, SEQ ID NO: 26) at varying concentrations. Luciferase measurements were performed after 24 hours.

Results:

Transfection of the 8-mer LNA-antimiR Compound 3221 (SEQ ID NO: 19) against miR-106b resulted in dose-dependent inhibition of miR-106b as shown by de-repression of the luciferase reporter, which was completely de-repressed at 1 nM LNA-antimiR concentration (FIG. 15). Comparable results were obtained using the 15-mer LNA-antimiR Compound 3228 (SEQ ID NO: 26) demonstrating that an 8-mer LNA-antimiR is similarly potent to a 15-mer.

Conclusion:

Targeting of miR-106b in HeLa cells shows that an 8-mer fully LNA-modified and phosphorotiolated LNA-antimiR is equally potent compared with a 15-mer LNA/DNA mixmer LNA-antimiR.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

5.200 cells were seeded per well in a 96-well plate the day before transfection in order to achieve 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 57 ng miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector together with 0.14 μl Lipofectamine-2000 (Invitrogen) according to the manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 30 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24-well plates was put on an orbital shaker for 30 min. The cells were collected and transferred to eppendorf tubes and spinned at 10.000 rpm for 30 min after which luciferase measurements were performed according to the manufacturer's instructions (Promega).

Example 17 Antagonism of miR-19a by a Fully LNA-Modified 8-Mer (Compound 3222, SEQ ID NO: 20) LNA-antimiR

To further confirm that our approach of using short LNA-antimiRs can be readily adapted to targeting of other miRNAs we designed a fully LNA-modified 8-mer LNA-antimiR against microRNA-19a. A perfect match target site for miR-19a was cloned in the 3′UTR of the luciferase gene in the psiCHECK2 vector. The reporter plasmid was transfected into the human cervix carcinoma HeLa cell line together with a short LNA-antimiR (Compound 3222, SEQ ID NO: 20) or with a 15-mer LNA-antimiR (Compound 3229, SEQ ID NO: 27) targeting miR-19a at varying concentrations. Luciferase measurements were performed after 24 hours.

Results:

As shown in FIG. 16, transfection of the 15-mer LNA-antimiR Compound 3229 (SEQ ID NO: 27) into HeLa efficiently antagonizes miR-19a as demonstrated by complete de-repression at 1 nM LNA-antimiR concentration. By comparison, transfection of the 8-mer LNA-antimiR Compound 3222 (SEQ ID NO: 20) resulted in effective miR-19a antagonism already at 0.5 nM concentration, indicating that this 8-mer LNA-antimiR is at least equally potent compared with a 15-mer LNA-antimiR in HeLa cells.

Conclusion:

Targeting of miR-19a in HeLa cells shows that an 8-mer fully LNA-modified and phosphorothiolated LNA-antimiR is at least equally potent compared with a 15-mer LNA/DNA mixmer LNA-antimiR.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

5.200 cells were seeded per well in a 96-well plate the day before transfection in order to achieve 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 57 ng miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector together with 0.14 μl Lipofectamine-2000 (Invitrogen) according to manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 30 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24-well plates was put on an orbital shaker for 30 min. The cells were collected and transferred to eppendorf tubes and spinned at 10.000 rpm for 30 min after which luciferase measurements were performed according to the manufacturer's instructions (Promega).

Example 18 Targeting of a microRNA Family Using Short, Fully LNA-Substituted LNA-antimiR

Next, we investigated whether it is possible to target a microRNA family using a single short 7-mer LNA-antimiR complementary to the seed sequence that is common for all family members (see FIG. 17). In this experiment, we focused on miR-221 and miR-222 that are overexpressed in solid tumors of the colon, pancreas, prostate and stomach. It has also been shown that miR-221 and miR-222 are the most significantly upregulated microRNAs in glioblastoma multiforme. Furthermore, overexpression of miR-221 and miR-222 may contribute to the growth and progression of prostate carcinoma, at least in part by blocking the tumor suppressor protein p27. A perfect match target site for both miR-221 and miR-222, respectively, was cloned into the 3′UTR of the luciferase gene resulting in two reporter constructs. These constructs were then transfected either separate or combined into the prostate carcinoma cell line, PC3. In addition to the 7-mer, targeting both miR-221 and miR-222, we also co-transfected a 15-mer LNA-antimiR (15mer) targeting either miR-221 (Compound 3223, SEQ ID NO: 21) or miR-222 (Compound 3224, SEQ ID NO: 22), each transfected separately or together (see FIG. 18 left).

Results:

As shown in FIG. 18, transfection of PC3 cells with the LNA-antimiR Compound 3223 (SEQ ID NO: 21) against miR-221 resulted in efficient inhibition of miR-221 at 1 nM LNA-antimiR concentration. An inhibitory effect is also observed when using the luciferase reporter plasmid for miR-222 as well as when co-transfecting both luciferase reporters for miR-221 and miR-222 simultaneously into PC3 cells. This inhibitory effect is most likely due to the shared seed sequence between miR-221 and miR-222. Similarly, transfection of PC3 cells with the LNA-antimiR Compound 3224 (SEQ ID NO: 22) against miR-222 resulted in efficient inhibition of miR-222 at 1 nM LNA-antimiR concentration as shown by complete de-repression of the luciferase reporter for miR-222. An inhibitory effect is also observed when using the luciferase reporter plasmid for miR-222 as well as when co-transfecting both luciferase reporters for miR-221 and miR-222 simultaneously into PC3 cells. Co-transfection of both LNA-antimiR compounds Compound 3223 (SEQ ID NO: 21) and Compound 3224 (SEQ ID NO: 22) against miR-221 and miR-222, respectively, (see FIG. 18 left), resulted in effective inhibition of both miRNAs as shown by complete de-repression of the luciferase reporter plasmids both when separately transfected and when co-transfected into PC3 cells. Interestingly, transfection of a single fully LNA-modified 7-mer LNA-antimiR (Compound 3225, SEQ ID NO: 23) targeting the seed sequence of miR-221 and miR-222 into PC3 cells resulted in efficient, dose-dependent antagonism of miR-221 and miR-222 simultaneously as shown by complete de-repression of the luciferase reporter plasmids both when separately transfected and when co-transfected into PC3 cells. This demonstrates that a single, short LNA-substituted LNA-antimiR can effectively target seed sequences thereby antagonizing entire microRNA families simultaneously. Analysis of the miRBase microRNA sequence database showed that the miR-221/222 seed recognition sequence, of the LNA-antimiR Compound 3225 (SEQ ID NO: 23) is unique for both miRNAs.

Conclusion:

Our results demonstrate that LNA enables design and synthesis of short fully LNA-substituted LNA-antimiR oligonucleotides that can effectively target microRNA seed sequences thereby antagonizing entire microRNA families simultaneously.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC (#90112714) PC3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

100.000 cells were seeded per well in a 12-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, PC3 cells were transfected with 0.3 ug of luciferase reporter plasmid for miR-221 or for miR-222 or with empty psiCHECK2 vector without miRNA target site as control together with 1.2 μl Lipofectamine-2000 (Invitrogen) according to the manufacturer's instructions. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 250 μl 1× Passive Lysis Buffer (Promega) was added to the wells. The plates were placed on a shaker for 30 min., after which the cell lysates was transferred to eppendorf tubes. The cell lysate was spinned for 10 min at 2.500 rpm after which 20 μl were transferred to a 96-well plate and luciferase measurements were performed according to the manufacturer's instructions (Promega).

Example 19 Assessment of p27 Protein Levels as a Functional Readout for Antagonism of the miR-221/222 Family by the 7-Mer Compound 3225 (SEQ ID NO: 23) LNA-antimiR

Previous work has shown (le Sage et al. 2007, Galardi et al. 2007) that miR-221 and miR-222 post-transcriptionally regulate the expression of the tumour suppressor gene p27, which is involved in cell cycle regulation. In these studies, down-regulation of miR-221 and miR-222 was shown to increase expression levels of p27. Thus, as a functional readout for antagonism of the miR-221/222 family by the 7-mer Compound 3225 (SEQ ID NO: 23) LNA-antimiR we determined the protein levels of p27 after transfection of the LNA-antimiR Compound 3225 (SEQ ID NO: 23) into PC3 cells in comparison with an 8-mer LNA mismatch control. After 24 hours the cells were harvested for western blot analysis (FIG. 19).

Results:

As shown in FIG. 19, transfection of the 7-mer LNA-antimiR Compound 3225 (SEQ ID NO: 23) targeting the seed sequence in miR-221 and miR-222 resulted in dose-dependent increase of the p27 protein levels compared to either untransfected or LNA mismatch control transfected PC3 cells. These results clearly demonstrate that the 7-mer LNA-antimiR is able to effectively antagonize the miR-221/222 family leading to de-repression of the direct target p27 at the protein level.

Conclusion:

A fully LNA-modified 7-mer LNA-antimiR targeting the seed sequence in the miR-221/222 family effectively antagonized both miRNAs leading to de-repression of the direct target p27 at the protein level.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC (#90112714) PC3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

250.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, PC3 cells were transfected with LNA-antimiRs at varying concentrations with Lipofectamine-2000. Cells were harvested after 24 hours for protein extraction and western blot analysis.

Western Blot:

Cells were washed with PBS, trypsinated, transferred to eppendorf tubes and 250 μl lysis buffer (1×RIPA) was added. The cell lysate was placed on ice for 20 min, then spinned at 10.000 rpm for 10 minutes. The protein concentration was measured with Coomassie Plus according to the manufacturer's instructions and 100 ug was loaded onto a 4-12% BIS-TRIS gel. The membrane was incubated overnight at 4° C. with the primary monoclonal mouse antibody p27 (BD Biosciences) at a 1:1000 dilution. Immunoreactive bands were visualized with ECL Plus (Amersham).

Example 20 Duplex Melting Temperatures (T_(m)) of the LNA-antimiRs

As shown in Table 5, T_(m) values increase with increasing the length of short fully modified LNA-antimiRs (see T_(m) values for Compound 3205 (SEQ ID NO: 2) and Compounds 3209 to 3214 (SEQ ID NOs: 7 to 12) in Table 7). Most optimal inhibitory effect was achieved with the 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) against miR-21, whereas the very low Tm of the 6-mer Compound 3209 (SEQ ID NO: 7) is most likely not sufficient to mediate antagonism of the miR-21 target. On the other hand, increasing the length beyond a 10-mer (Compound 3212, SEQ ID NO: 10) significantly increases the T_(m) while simultaneously decreasing the inhibitory activity as measured using the luciferase miR-21 reporter, which is most likely due to high propensity of the fully modified 12- and 14-mer LNA-antimiRs to form homodimers. The experiments using a sliding window of fully LNA-modified 8-mer LNA-antimirs across the mir-21 recognition sequence clearly demonstrate that in addition to adequate T_(m) value of the LNA-antimiR, the seed region is most critical for miRNA function and, thus, the most optimal region to be targeted by an LNA-antimiR.

TABLE 5 T_(m) values for miR-21 LNA-antimiRs, measured against a complementary RNA oligonucleotide Measured SEQ Length T_(m )(RNA) Compound ID NO: microRNA (bp) Sequence  ° C. 3205 2 miR-21 8 5′-GATAAGCT-3′ 64.0 3209 7 miR-21 6 5′-TAAGCT-3′ 32.0 3210 8 miR-21 7 5′-ATAAGCT-3′ 45.0 3211 9 miR-21 9 5′-TGATAAGCT-3′ 65.0 3212 10 miR-21 10 5′-CTGATAAGCT-3′ 63.0 3213 11 miR-21 12 5′-GTCTGATAAGCT-3′ 86.8 3214 12 miR-21 14 5′-CAGTCTGATAAGCT-3′ 89.9 3215 13 miR-21 8 5′-TCTGATAA-3′ 56.0 3216 14 miR-21 8 5′-ATCAGTCT-3 72.0 3217 15 miR-21 8 5′-TCAACATC-3 48.0 Conclusion:

The T_(m) values along with experimental data obtained with luciferase reporters show that potent antagonism by LNA-antimiR is not only dependent on T_(m) but also depends on the positioning of the LNA-antimiR within the microRNA recognition sequence.

Materials and Methods:

T_(m) Measurements:

The oligonucleotide:miR-21 RNA duplexes were diluted to 3 μM in 500 μl RNase free H₂0 and mixed with 500 μl 2× T_(m)-buffer (200 mM NaCl, 0.2 mM EDTA, 20 mM Na-phosphate, pH 7.0). The solution was heated to 95° C. for 3 min and then allowed to anneal in RT for 30 min. The duplex melting temperatures (T_(m)) were measured on a Lambda 40 UV/VIS Spectrophotometer equipped with a Peltier temperature programmer PTP6 using PE Templab software (Perkin Elmer). The temperature was ramped up from 20° C. to 95° C. and then down to 25° C., recording absorption at 260 nm. First derivative and the local maximums of both the melting and annealing were used to assess the duplex melting temperatures.

Example 21 Assessment of miR-21 Antagonism by Comparing an 8-Mer (Compound 3205, SEQ ID NO: 2) Versus a 15-Mer (Compound 3204, SEQ ID NO: 1) LNA-antimiR in Human Hepatocytic Cell Line HepG2

We have previously shown in this application, that an 8-mer LNA-antimiR that is fully LNA-modified and phosphorothiolated effectively antagonizes miR-21 in the human cervix carcinoma cell line HeLa, the human breast carcinoma cell line MCF-7 and the human prostate cancer cell line PC3. We extended this screening approach to the human hepatocellular cancer cell line HepG2. To assess the efficiency of the 8-mer LNA-antimiR oligonucleotide against miR-21, luciferase reporter constructs were generated in which a perfect match target site for the mature miR-21 was cloned into the 3′UTR of the Renilla luciferase gene. In order to monitor miR-21 inhibition, HepG2 cells were transfected with the luciferase constructs together with the miR-21 antagonist Compound 3205 (SEQ ID NO: 2) (8-mer) and for comparison of specificity with the 8-mer LNA-antimiR mismatch (Compound 3218, SEQ ID NO: 16) and for comparison of potency together with the 15-mer (Compound 3204, SEQ ID NO: 1) at varying concentrations. After 24 hours, luciferase activity was measured.

Results:

The luciferase reporter experiments showed a dose-dependent de-repression of the luciferase miR-21 reporter activity with the 15-mer LNA-antimiR against miR-21 (Compound 3204, SEQ ID NO: 1). However, complete de-repression of the luciferase reporter was not obtained, not even at the higher concentrations (FIG. 20). In contrast, the cells that were transfected with the 8-mer fully LNA modified LNA-antimiR (Compound 3205, SEQ ID NO: 2) showed complete de-repression already at 5 nM, indicating significantly improved potency compared to the 15-mer LNA-antimiR. Comparing the specificity of the 8-mer perfect match and the 8-mer mismatch, the mismatch LNA-antimiR (Compound 3218, SEQ ID NO: 16) did not show any de-repression at all, demonstrating high specificity of the LNA-antimiR compound against miR-21.

Conclusion:

The 8-mer (Compound 3205, SEQ ID NO: 2) is more potent than the 15-mer LNA-antimiR in targeting miR-21 and antagonism of miR-21 by Compound 3205 (SEQ ID NO: 2) is specific.

Materials and Methods:

Cell Line:

The human hepatocytic HepG2 cell line was purchased from ECACC (#85011430). HepG2 cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

650.000 cells were seeded per well in a 6-well plate and reverse transfection were performed. HepG2 cells were transfected with 0.6 μg miR-21 or empty psiCHECK2 vector together with 2.55 μl Lipofectamine-2000 (Invitrogen) according to manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 300 μl 1× Passive Lysis Buffer (Promega) was added to the wells. The plates were placed on a shaker for 30 min., after which the cell lysates were transferred to eppendorf tubes. The cell lysate was centrifugated for 10 min at 2.500 rpm after which 50 μl were transferred to a 96 well plate and luciferase measurements were performed according to the manufacturer's instructions (Promega).

Example 22 Validation of Interaction of the miR-21 Target Site in the Pdcd4 3′UTR and miR-21 Using the 8-Mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR in Human Hepatocellular Cell Line Huh-7

The tumour suppressor protein Pdcd4 inhibits tumour promotion and progression. Furthermore, downregulation of Pdcd4 in lung and colorectal cancer has also been associated with poor patient prognosis. Recently, Asangani et al (Oncogene 2007) and Frankel et al (J Biol Chem 2008) showed that the Pdcd4 3′UTR contains a conserved target site for miR-21, and transfecting cells with an antimiR-21, resulted in an increase in Pdcd4 protein. We therefore constructed a luciferase reporter plasmid, harboring 313 nt of the 3′UTR region of Pdcd4 encompassing the aforementioned miR-21 target site, which was co-transfected together with different LNA-antimiRs and pre-miR-21 (10 nM) into Huh-7 cells. The different LNA-antimiRs were; Compound 3205 (SEQ ID NO: 2) (8-mer, perfect match), Compound 3218 (SEQ ID NO: 16) (8-mer, mismatch) and Compound 3204 (SEQ ID NO: 1) (15-mer, DNA/LNA mixmer). Luciferase measurements were performed after 24 hours.

Results:

As shown in FIG. 21, cells transfected with the Pdcd4 3′UTR luciferase reporter and Compound 3205 (SEQ ID NO: 2), an increase in luciferase activity was observed, indicating interaction between the Pdcd4 3′UTR and miR-21. However, transfecting the cells with the mismatch compound, Compound 3218 (SEQ ID NO: 16), no change in luciferase activity was observed, which was expected since the compound does not antagonize miR-21. When comparing the 8-mer LNA-antimiR against the 15-mer LNA-antimiR (Compound 3204, SEQ ID NO: 1), the short fully LNA-modified and phosphorothiolated LNA-antimiR was significantly more potent, confirming previous data.

Materials and Methods:

Cell Line:

The human hepatoma cell line Huh-7 was a kind gift from R. Bartinschlager (Dept Mol Virology, University of Heidelberg). Huh-7 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

11.000 cells were seeded per well in a 96-well plate the day before transfection in order to achieve 50-70% confluency the next day. On the day of transfection, Huh-7 cells were transfected with 20 ng Pdcd4 3′UTR/psiCHECK2 or empty psiCHECK2 vector together with 10 nM pre-miR-21 (Ambion) and 0.14 μl Lipofectamine-2000 (Invitrogen) according to the manufacturer's instructions. Varying concentrations of the LNA-antimiR oligonucleotides were also transfected. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

Cells were washed and 30 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 96-well plates was put on an orbital shaker. After 30 min., 50 μl luciferase substrate dissolved in Luciferase Assay Buffer II (Dual-Luciferase Reporter Assay System from Promega, Cat# E1910) was added to the wells with lysated cells and luciferase measurements were performed according to the manufacturer's instructions (Promega).

Example 23 Assessment of Pdcd4 Protein Levels as a Functional Readout for miR-21 Antagonism by the 8-Mer LNA-antimiR (Compound 3205, SEQ ID NO: 2)

In addition, we also transfected HeLa cells with Compound 3205 (SEQ ID NO: 2) (perfect match), Compound 3218 (SEQ ID NO: 16) (mismatch), Compound 3219 (SEQ ID NO: 17) (scrambled) and analyzed Pdcd4 protein levels after 24 hours with Western blot (FIG. 22). As shown, in the protein extracts from cells where Compound 3205 (SEQ ID NO: 2) had been added, the Pdcd4 protein levels increase, due to antagonism of mir-21 by Compound 3205 (SEQ ID NO: 2) in contrast to the two control LNA oligonucleotides.

Conclusion:

Antagonism of miR-21 using an 8-mer (Compound 3205, SEQ ID NO: 2) leads to derepression of the direct target Pdcd4□ntagorism of miR-21

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

200.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 5 nM LNA oligonucleotides and 2.5 μg/ml Lipofectamine-2000 (Invitrogen) according to the manufacturer's instructions. After 24 hours, cells were harvested for Western blot analysis.

Western Blot:

Cells were washed with PBS, trypsinated, transferred to eppendorf tubes and 50 μl lysis buffer (1×RIPA) was added. The cell lysate was placed on ice for 20 min and spinned at 10.000 rpm for 10 minutes. Equal amounts (15 μl cell lysate) were loaded onto a 4-12% BIS-TRIS gel. The proteins were transferred to a nitrocellulose membrane using iBlot (Invitrogen) according to manufacturers instructions. The membrane was incubated overnight at 4° C. with the primary affinity purified rabbit serum antibody Pdcd4 (Rockland) with a 1:2000 concentration. As control, anti-beta tubulin antibodies (Thermo Scientific) were used at a 1:5000 dilution. Immunoreactive bands were visualized with ECL Plus (Amersham).

Example 24 Assessment of Potential Hepatotoxicity of the 8-Mer Perfect Match LNA-antimiR Compound 3205 (SEQ ID NO: 2) and the LNA Mismatch Control Compound 3218 (SEQ ID NO: 16)

Each compound was injected into female NMRI mice, at doses of 25 mg/kg, 5 mg/kg and 1 mg/kg, every other day for 2 weeks. The animals were sacrificed and serum was collected from whole blood for ALT and AST analyses. As seen in FIG. 23, the ALT and AST levels were not elevated for Compound 3205 (SEQ ID NO: 2) compared to saline or Compound 3218 (SEQ ID NO: 16) (mismatch control). However, one mouse showed increased levels (marked red), since the serum samples were contaminated with red blood cells, which contain 6-8 times higher levels of ALT and AST compared to plasma. The mice that received 5 mg/kg and 1 mg/kg were also analyzed for ALT and AST levels and showed no changes compared to saline treated control animals (data not shown).

Materials and Methods:

Experimental Design:

Conc. Gr. Animal No. of Compound at dose vol. no. IDno. mice Dose level per day 10 ml/mg Adm. Route Dosing 1  1-10 10 NaCl — i.v 0, 2, 4, 7, 9 0.9% 2 11-15 5 Compound 3205 2.5 mg/ml i.v 0, 2, 4, 7, 9 (SEQ ID NO: 2) 25 mg/kg  3 16-20 5 Compound 3205 0.5 mg/ml i.v 0, 2, 4, 7, 9 (SEQ ID NO: 2) 5 mg/kg 4 21-25 5 Compound 3205 0.1 mg/ml i.v 0, 2, 4, 7, 9 (SEQ ID NO: 2) 1 mg/kg 5 26-30 5 Compound 3230 2.5 mg/ml i.v 0, 2, 4, 7, 9 (SEQ ID NO: 16) 25 mg/kg 6 31-35 5 Compound 3230 0.5 mg/ml i.v 0, 2, 4, 7, 9 (SEQ ID NO: 16) 5 mg/kg Sacrifice;

The animals was sacrificed by cervical dislocation.

Sampling of Serum for ALT/AST;

The animals were anaesthetised with 70% CO₂-30% O₂ before collection of retro orbital sinus blood. The blood was collected into S-monovette Serum-Gel vials. The serum samples were harvested and stored from each individual mouse. The blood samples were stored at room temperature for two hours and thereafter centrifuged 10 min, 3000 rpm, at room temp. The serum fractions were harvested into Eppendorf tubes on wet ice.

ALT and AST Measurements;

ALT and AST measurements were performed in 96-well plates using ALT and AST reagents from ABX Pentra (A11A01627-ALT, A11A01629-AST) according to the manufacturer's instructions. In short, serum samples were diluted 2.5 fold with H₂O and each sample was assayed in duplicate. After addition of 50 μl diluted sample or standard (multical from ABX Pentra—A11A01652) to each well, 200 μl of 37° C. AST or ALT reagent mix was added to each well. Kinetic measurements were performed for 5 min with an interval of 30s at 340 nm and 37° C.

Example 25 Assessment of PU.1 Protein Levels as a Functional Readout for miR-155 Antagonism by Short LNA-antimiR (Compound 3207, SEQ ID NO: 4)

We have previously shown that the 8-mer (Compound 3207, SEQ ID NO: 4) antagonizing miR-155 leads to derepression of the miR-155 target c/EBPbeta in the mouse macrophage RAW cells. To further verify the potency of Compound 3207 (SEQ ID NO: 4) we determined the protein levels of another miR-155 target, PU.1 As a functional readout for miR-155 antagonism by short LNA-antimiR (Compound 3207, SEQ ID NO: 4) we performed Western blot. The antagonism was verified in the human monocytic THP-1 cell line which was transfected together with either an 8-mer (Compound 3207, SEQ ID NO: 4) perfect match or a 8-mer control LNA in the absence or presence of pre-miR-155. LPS was used to induce miR-155 accumulation and cells were harvested after 24 hours.

Results:

THP-1 cells that were transfected with pre-miR-155 shows a decrease in PU.1 levels (FIG. 24). Transfecting the cells with the fully LNA-modified and phosphorothiolated Compound 3207 (SEQ ID NO: 4) effectively antagonizes miR-155, leading to unaltered levels of PU.1 protein. By comparison, transfecting the cells with an 8-mer LNA control, PU.1 levels decreased, indicating that antagonism of miR-155 by Compound 3207 (SEQ ID NO: 4) LNA-antimiR is specific.

Conclusion:

Antagonism of miR-155 using an 8-mer leads to de-repression of the direct target PU.1 in human THP-1 cells.

Materials and Methods:

Cell Line:

The human monocytic THP-1 cell line was purchased from ECACC (#88081201). THP-1 cells were cultured in RPMI with L-glutamine, supplemented with 10% fetal bovine serum.

Transfection:

200.000 cells were seeded per well in a 12-well plate the day before. On the day of transfection, THP-1 cells were transfected with 5 nmol pre-miR-155 (Ambion) and/or 5 nM LNA-antimiR together with Lipofectamine-2000 (Invitrogen) according to the manufacturer's instructions. LPS (100 ng/ml) was added to the cells after the 4 hour incubation with the transfection complexes. After 24 hours, cells were harvested for protein extraction and western blot analysis.

Western Blot:

Cells were washed with PBS, trypsinated, transferred to eppendorf tubes and 50 μl lysis buffer (1×RIPA) was added. The cell lysate was placed on ice for 20 min and spinned at 10.000 rpm for 10 minutes. Equal amounts (15 μl cell lysate) were loaded onto a 4-12% BIS-TRIS gel. The proteins were transferred to a nitrocellulose membrane using iBlot (Invitrogen) according to manufacturers instructions The membrane was incubated overnight at 4° C. with the rabbit monoclonal PU.1 antibody (Cell Signaling) with a 1:2000 concentration. As equal loading, Tubulin (Thermo Scientific) was used at a 1:5000 dilution. Immunoreactive bands were visualized with ECL Plus (Amersham).

Example 26 Assessment of p27 Protein Levels as a Functional Readout for Antagonism of the miR-221/222 Family by the 7-Mer Compound 3225 (SEQ ID NO: 23) LNA-antimiR

Previous work has shown (le Sage et al. 2007, Galardi et al. 2007) that miR-221 and miR-222 post-transcriptionally regulate the expression of the tumour suppressor gene p27, which is involved in cell cycle regulation. In these studies, down-regulation of miR-221 and miR-222 was shown to increase expression levels of p27. Thus, as a functional readout for antagonism of the miR-221/222 family by the 7-mer Compound 3225 (SEQ ID NO: 23) LNA-antimiR we determined the protein levels of p27 after transfection of the LNA-antimiR Compound 3225 (SEQ ID NO: 23) into PC3 cells.

Results:

As shown in FIG. 25, transfection of the 7-mer LNA-antimiR Compound 3225 (SEQ ID NO: 23) targeting the seed sequence of miR-221 and miR-222 resulted in dose-dependent increase of the p27 protein levels compared to either untransfected or our LNA scrambled control transfected PC3 cells. These results clearly demonstrate that the 7-mer LNA-antimiR is able to effectively antagonize the miR-221/222 family leading to de-repression of the direct target p27 at the protein level at concentrations as low as 5 nM.

Conclusion:

A fully LNA-modified 7-mer LNA-antimiR targeting the seed sequence in the miR-221/222 family at 5 nM can effectively antagonize both miRNAs leading to de-repression of the direct target p27 at protein level.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC (#90112714). PC3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

250.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, PC3 cells were transfected with LNA-oligonucleotides at varying concentrations (see FIG. 25) with Lipofectamine-2000. Cells were harvested after 24 hours for protein extraction and western blot analysis.

Western Blot:

Cells were washed with PBS, trypsinated, transferred to eppendorf tubes and 50 μl lysis buffer (1×RIPA) was added. The cell lysate was placed on ice for 20 min, then spinned at 10.000 rpm for 10 minutes. Equal amounts (15 μl cell lysate) were loaded onto a 4-12% BIS-TRIS gel. The proteins were transferred to a nitrocellulose membrane using iBlot (Invitrogen) according to manufacturers instructions. The membrane was incubated overnight at 4° C. with the primary monoclonal mouse antibody p27 (BD Biosciences) at a 1:1000 dilution. As loading control, Tubulin (Thermo Scientific) was used at a 1:5000 dilution. Immunoreactive bands were visualized with ECL Plus (Amersham).

Example 27 Knock-Down of miR-221/222 by the 7-Mer Compound 3225 (SEQ ID NO: 23) LNA-antimiR Reduces Colony Formation of PC3 Cells

A hallmark of cellular transformation is the ability for tumour cells to grow in an anchorage-independent way in semisolid medium. We have therefore performed soft agar assay which is a phenotypic assay that is relevant for cancer, given that it measures the decrease of tumour cells. We transfected Compound 3225 (SEQ ID NO: 23) (perfect match) and Compound 3231 (SEQ ID NO: 28) (scrambled) into PC3 cells, and after 24 hours plated cells in soft agar. Colonies were counted after 12 days. We show in FIG. 26 that inhibition of miR-221 and miR-222 by Compound 3225 (SEQ ID NO: 23) can reduce the amount of colonies growing in soft agar compared to the scrambled control LNA-antimiR, indicating decrease of tumour cells.

Conclusion:

The 7-mer (Compound 3225, SEQ ID NO: 23) targeting the miR-221/222 family reduces the number of colonies in soft agar, indicating proliferation arrest of PC3 cells.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC (#90112714). PC3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

250.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, PC3 cells were transfected with 25 nM of different LNA oligonucleotides with Lipofectamine-2000.

Clonogenic Growth in Soft Agar:

2.5×10³ PC3 cells were seeded in 0.35% agar on the top of a base layer containing 0.5% agar. Cells were plated 24 hours after transfection. Plates were incubated in at 37° C., 5% CO₂ in a humified incubator for 12 days and stained with 0.005% crystal violet for 1 h, after which cells were counted. The assay was performed in triplicate.

Example 28 Assessment of Let-7 Antagonism by 6-9-Mer LNA-antimiRs in Huh-7 Cells Transfected with Let-7a Precursor miRNA, and a Luciferase Sensor Assay

In order to assess the efficiency of fully LNA-modified 6-9-mer oligonucleotides in targeting and antagonizing the let-7 family of miRNAs, a luciferase sensor construct was made, containing some 800 bp of the HMGA2 3′UTR. The sequence cloned into the vector contains four out of seven functional let-7 binding sites (sites 2-5), as previously demonstrated by Mayr et al. (Science, 2007) and Lee and Dutta (Genes Dev, 2007). In order to monitor let-7 inhibition, the hepatocellular carcinoma cell line Huh-7 (with low to non-existing levels of endogenous let-7) was transfected with the luciferase sensor construct, with let-7a precursor miRNA, and with the 6-9 mer let-7 antagonists Compound 3232 (SEQ ID NO: 29), Compound 3233 (SEQ ID NO: 30), Compound 3227 (SEQ ID NO: 25), Compound 3234 (SEQ ID NO: 31), Compound 3235 (SEQ ID NO: 32); see FIG. 27) at increasing concentrations. The 6-9-mer LNA-antimiRs were compared with Compound 3226 (SEQ ID NO: 33), a 15-mer against let-7a as a positive control. After 24 hours, luciferase activity was measured.

Results:

As seen in FIG. 28, the fully LNA-modified 8- and 9-mer LNA-antimiRs (Compound 3227 (SEQ ID NO: 34), Compound 3234 (SEQ ID NO: 31), and Compound 3235 (SEQ ID NO: 32)) show similar potencies in de-repressing the let-7 targets in the luciferase sensor assay, as the positive control 15-mer Compound 3226 (SEQ ID NO: 24). Full target de-repression for these highly potent compounds is achieved already at 1-5 nM, whereas the 7-mer Compound 3233 (SEQ ID NO: 30) needs to be present at slightly higher concentrations (10 nM) to generate the same effect. However, the 6-mer Compound 3232 (SEQ ID NO: 29) shows no effect even at as high concentrations as 50 nM. The de-repression of luciferase activity by the 7-9- and the 15-mer LNA-antimiRs is dose-dependent, which is particularly clear in the case of the slightly less potent Compound 3233 (SEQ ID NO: 30).

Conclusion:

To conclude, the 8-9-mer LNA-antimiRs (Compound 3227 (SEQ ID NO: 25), Compound 3234 (SEQ ID NO: 31), and Compound 3235 (SEQ ID NO: 32)) show equal antagonist potencies in inhibition of let-7a in vitro compared to the 15-mer LNA-antimiR Compound 3226 (SEQ ID NO: 24) targeting let-7a. A potent effect, albeit at slightly higher concentrations is also seen for the 7-mer Compound 3233 (SEQ ID NO: 30), whereas a 6-mer has no effect at tested concentrations.

Materials and Methods:

Cell Line:

The hepatocellular carcinoma cell line Huh-7 was a kind gift from R. Bartinschlager (Dept Mol Virology, University of Heidelberg).Huh-7 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

8,000 cells were seeded per well in a 96-well plate the day before transfection in order to receive 60-80% confluency the next day. On the day of transfection, Huh-7 cells in each well were transfected with 20 ng HMGA2 3′UTR/psiCHECK2 plasmid, let-7a precursor miRNA (Dharmacon; 10 nM end-concentration), LNA-antimiRs Compound 3232 (SEQ ID NO: 29), Compound 3233 (SEQ ID NO: 30), Compound 3227 (SEQ ID NO: 25), Compound 3234 (SEQ ID NO: 31), Compound 3235 (SEQ ID NO: 32), Compound 3226 (SEQ ID NO: 24); 0-50 nM end concentrations) together with 0.17 μl Lipofectamine-2000 (Invitrogen) according to manufacturer's instructions. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

Growth media was discarded and 30 μl 1× Passive Lysis Buffer (Promega) was added to each well. After 15-30 minutes of incubation on an orbital shaker, renilla and firefly luciferase measurements were performed according to manufacturer's instructions (Promega).

Example 29 Assessment of Entire Let-7 Family Antagonism by 8-, and 15-Mer LNA-antimiRs in Huh-7 Cells Transfected with a Luciferase Sensor Assay

In order to assess the efficiency of a fully LNA-modified 8-mer oligonucleotide in antagonizing the entire let-7 family of miRNAs, the same luciferase sensor construct as described in the previous example was used. Again, Huh-7 cells (with low to non-existing levels of endogenous let-7) were transfected with the sensor construct, with one of the family-representative let-7a, let-7d, let-7e, or let-7i precursors, and with the antagonist Compound 3227 (SEQ ID NO: 25) at increasing concentrations. The 8-mer LNA-antimiR was compared to Compound 3226 (SEQ ID NO: 24), a 15-mer against let-7a as a positive and potent control. After 24 hours, luciferase activity was measured.

Results:

As seen in FIG. 29 the fully LNA-modified 8-mer LNA-antimiRs (Compound 3227) (SEQ ID NO: 25) show similar potencies in de-repressing the various let-7 targets in the luciferase sensor assay, as the positive control 15-mer Compound 3226 (SEQ ID NO: 24). Nearly full target de-repression for the 8-mer is achieved already at 0.5-1 nM, except in the case with let-7e premiR (FIG. 29C), to which only 7 out of 8 nucleotides of Compound 3227 (SEQ ID NO: 25) hybridizes to the target. However, despite the terminal mismatch in this case, Compound 3227 (SEQ ID NO: 25) generates full target de-repression at 5 nM. The positive control 15-mer shows potent antagonism of all precursors and gives nearly full de-repression at 0.5 nM. The de-repression of luciferase activity by both the 8- and the 15-mer LNA-antimiRs is clearly dose-dependent, as seen in all four panels (FIG. 29A-D).

Conclusion:

To conclude, the 8-mer LNA-antimiR (Compound 3227, SEQ ID NO: 25), is a potent antagonist against four representative let-7 family members in vitro, and thus likely against the entire family. Compared to a 15-mer positive control antagonist, Compound 3226 (SEQ ID NO: 24), the 8-mer is equally potent for three of four targets, and slightly less potent for the fourth target, let-7e, explained by a terminal mismatch in this case.

Materials and Methods:

Cell Line:

The hepatocellular carcinoma cell line Huh-7 was a kind gift from R. Bartinschlager (Dept Mol Virology, University of Heidelberg). Huh-7 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

8,000 cells were seeded per well in a 96-well plate the day before transfection in order to receive 60-80% confluency the next day. On the day of transfection, Huh-7 cells in each well were transfected with 20 ng HMGA2 3′UTR/psiCHECK2 plasmid, with let-7a, -7d, -7e, or -7i precursor miRNA (Dharmacon; 10 nM end-concentration), and with LNA-antimiRs Compound 3227 (SEQ ID NO: 25) and Compound 3226 (SEQ ID NO: 24); 0-50 nM end concentrations) together with 0.17 μl Lipofectamine-2000 (Invitrogen) according to manufacturer's instructions. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

Growth medium was discarded and 30 μl 1× Passive Lysis Buffer (Promega) was added to each well. After 15-30 minutes of incubation on an orbital shaker, renilla and firefly luciferase measurements were performed according to manufacturer's instructions (Promega).

Example 30 Assessment of Endogenous Let-7 Antagonism by Compound 3227 (SEQ ID NO: 25), an 8-mer LNA-antimiRs, in HeLa Cells Transfected with a Luciferase Sensor Assay

In order to determine the efficiency of a fully LNA-modified 8-mer oligonucleotide in targeting and antagonizing endogenous let-7, the same luciferase sensor construct as described in previous two examples, was co-transfected with Compound 3227 (SEQ ID NO: 25) into the cervical cancer cell line HeLa (that expresses moderate to high levels of let-7 as determined by Q-PCR; data not shown). Empty psiCHECK-2 vector was included as a negative control.

Results:

As seen in FIG. 30, the fully LNA-modified 8-mer LNA-antimiR Compound 3227 (SEQ ID NO: 25) shows potent antagonism of endogenous let-7, and gives full target de-repression at concentrations of 5-10 nM. The de-repression of luciferase activity is dose-dependent, starting around 1 nM and reaching a plateau at approximately 10 nM.

Conclusion:

To conclude, the 8-mer LNA-antimiR (Compound 3227, SEQ ID NO: 25), is a potent antagonist against also endogenous let-7 in vitro, and thus provides definite evidence that entire miRNA families can be successfully targeted by short and fully LNA-modified antagonists.

Materials and Methods:

Cell Line:

The cervical cancer cell line HeLa was purchased from ATCC (#CCL-2™). HeLa cells were cultured in Eagle's MEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

8,000 cells were seeded per well in a 96-well plate the day before transfection in order to receive 50-70% confluency the next day. On the day of transfection, HeLa cells in each well were co-transfected with 20 ng HMGA2 3′UTR/psiCHECK2 plasmid or psiCHECK-2 (empty vector), and with LNA-antimiR Compound 3227 (SEQ ID NO: 25) (0-50 nM, end concentrations) together with 0.17 μl Lipofectamine-2000 (Invitrogen) according to manufacturer's instructions. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

Growth media was discarded and 30 μl 1× Passive Lysis Buffer (Promega) was added to each well. After 15-30 minutes of incubation on an orbital shaker, renilla and firefly luciferase measurements were performed according to manufacturer's instructions (Promega).

Example 31 Assessment of miR-21 Antagonism by an 8-Mer LNA-antimiR-21 (Compound 3205, SEQ ID NO: 2) Versus an 8-Mer (Compound 3219, SEQ ID NO: 17) Scrambled Control LNA in the Human Colon Carcinoma Cell Line HCT116

We have previously shown in this application, that an 8-mer LNA-antimiR that is fully LNA-modified and phosphorothiolated effectively antagonizes miR-21 in the human cervix carcinoma cell line HeLa, the human breast carcinoma cell line MCF-7, the human prostate cancer cell line PC3 and human hepatocellular carcinoma HepG2 cell line. We extended this screening approach to the human colon carcinoma cell line HCT116. To assess the efficiency of the 8-mer LNA-antimiR oligonucleotide against miR-21, luciferase reporter constructs were generated in which a perfect match target site for the mature miR-21 was cloned into the 3′UTR of the Renilla luciferase gene. In order to monitor miR-21 inhibition, HCT116 cells were transfected with the luciferase constructs together with the miR-21 antagonist Compound 3205 (SEQ ID NO: 2) (8-mer) and for comparison of specificity with the 8-mer LNA scrambled control (Compound 3219, SEQ ID NO: 17). After 24 hours, luciferase activity was measured.

Results:

The luciferase reporter experiments showed a dose-dependent de-repression of the luciferase miR-21 reporter activity with the 8-mer LNA-antimiR against miR-21 (Compound 3205, SEQ ID NO: 2) and complete de-repression was obtained at 5 nM (FIG. 31). When comparing the specificity of the 8-mer perfect match and the 8-mer scrambled control, the scrambled control LNA-antimiR (Compound 3219, SEQ ID NO: 17) did not show any de-repression at all, demonstrating high specificity of the LNA-antimiR compound against miR-21.

Conclusion:

The 8-mer (Compound 3205, SEQ ID NO: 2) is potent in targeting miR-21 and antagonism of miR-21 by Compound 3205 (SEQ ID NO: 2) is specific.

Materials and Methods:

Cell Line:

The human colon carcinoma HCT116 cell line was purchased from ATCC(CCL-247). HCT116 cells were cultured in RPMI medium, supplemented with 10% fetal bovine serum, and 25 ug/ml Gentamicin.

Transfection:

110.000 cells were seeded per well in a 12-well plate and transfection was performed. HCT116 cells were transfected with 0.3 μg miR-21 luciferase sensor plasmid or empty psiCHECK2 vector together with 1.2 μl Lipofectamine-2000 (Invitrogen) according to the manufacturer's instructions. Transfected were also varying concentrations of LNA-antimiR and control oligonucleotides. After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 250 μl 1× Passive Lysis Buffer (Promega) was added to the wells. The plates were placed on a shaker for 30 min., after which the cell lysates were transferred to eppendorf tubes. The cell lysate was centrifugated for 10 min at 2.500 rpm after which 50 μl were transferred to a 96 well plate and luciferase measurements were performed according to the manufacturer's instructions (Promega).

Example 32 Knock-Down of miR-21 by the 8-Mer Compound 3205 (SEQ ID NO: 2) LNA-antimir Reduces Colony Formation of PC3 Cells

A hallmark of cellular transformation is the ability for tumour cells to grow in an anchorage-independent way in semisolid medium. We therefore performed soft agar assay which is a phenotypic assay that is relevant for cancer, given that it measures the decrease of tumour cells. We transfected Compound 3205 (SEQ ID NO: 2) (perfect match LNA-antimiR-21) and Compound 3219 (SEQ ID NO: 17) (LNA scrambled control) into PC3 cells, and after 24 hours plated cells in soft agar. Colonies were counted after 12 days. We show in FIG. 32 that inhibition of miR-21 by Compound 3205 (SEQ ID NO: 2) can reduce the amount of colonies growing in soft agar compared to the scrambled control LNA treated or untreated control (transfected, but with no LNA), demonstrating decrease of tumour cells.

Conclusion:

The 8-mer (Compound 3205, SEQ ID NO: 2) targeting the miR-21 family reduces the number of colonies in soft agar, demonstrating proliferation arrest of PC3 cells.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC (#90112714). PC3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

250.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, PC3 cells were transfected with 25 nM of different LNA oligonucleotides with Lipofectamine-2000.

Clonogenic Growth in Soft Agar:

2.5×10³ PC3 cells were seeded in 0.35% agar on the top of a base layer containing 0.5% agar. Cells were plated 24 hours after transfection. Plates were incubated in at 37° C., 5% CO₂ in a humified incubator for 12 days and stained with 0.005% crystal violet for 1 h, after which cells were counted. The assay was performed in triplicate.

Example 33 Silencing of miR-21 by the 8-Mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR Reduces Colony Formation of HepG2 Cells

miR-21 is overexpressed in the human hepatocellular carcinoma cell line HepG2 and we have previously shown that we are able to regulate the luciferase activity of a miR-21 sensor plasmid with Compound 3205 (SEQ ID NO: 2) in these cells. HepG2 cells were transfected with Compound 3205 (SEQ ID NO: 2) and Compound 3219 (SEQ ID NO: 17) (scrambled 8-mer), and after 24 hours plated into soft agar. Colonies were counted after 17 days with a microscope.

Results:

We show in FIG. 33 that inhibition of miR-21 by Compound 3205 (SEQ ID NO: 2) can reduce the amount of colonies growing in soft agar, showing that proliferation arrest has occurred. In addition, our scrambled 8-mer control, Compound 3219 (SEQ ID NO: 17), had no significant effect on the number of colonies.

Conclusion:

The 8-mer (Compound 3205, SEQ ID NO: 2) targeting the miR-21 reduces the number of colonies in soft agar, indicating proliferation arrest of HepG2 cells.

Materials and Methods:

Cell Line:

The human hepatocytic HepG2 cell line was purchased from ECACC (#85011430). HepG2 cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

650.000 cells were seeded per well in a 6-well plate and reverse transfection was performed. HepG2 cells were transfected with 0.6 μg miR-21 luciferase sensor plasmid or empty psiCHECK2 vector together with 2.55 μl Lipofectamine-2000 (Invitrogen) according to the manufacturer's instructions. Transfected were also LNA-antimiR and control oligonucleotides as varying concentrations. After 24 hours, the cells were harvested for luciferase measurements.

Clonogenic Growth in Soft Agar:

2.0×10³ HepG2 cells were seeded in 0.35% agar on the top of a base layer containing 0.5% agar. Cells were plated 24 hours after transfection. Plates were incubated in at 37° C., 5% CO₂ in a humified incubator for 17 days and stained with 0.005% crystal violet for 1 h, after which cells were counted. The assay was performed in triplicate.

Example 34 Silencing of miR-21 by the 8-Mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR Inhibits Cell Migration in PC3 Cells

Cell migration can be monitored by performing a wound healing assay (=scratch assay) where a “scratch” is made in a cell monolayer, and images are captured at the beginning and at regular intervals during cell migration. By comparing the images, quantification of the migration rate of the cells can be determined. This was done in the human prostate cancer cell line PC3. Cells were seeded, and on day 3 the cells were transfected, and the next day, when 100% confluency was reached, a scratch (=wound) was made. When the scratch was made, pictures were taken in order to document the initial wound. Afterwards the area of the wound closure is measured at different time points with the free software program Image J. As shown in FIG. 34A, PC3 cells had been treated with 25 nM Compound 3205 (SEQ ID NO: 2) (perfect match, miR-21), the control Compound 3219 (SEQ ID NO: 17) or left untransfected. Pictures were taken after 24 hours, and the area was calculated for the wound closure at respective time-point. The wound closure for the untransfected cells and for the control, Compound 3219 (SEQ ID NO: 17), was faster as compared to our LNA-antimiR against miR-21, Compound 3205 (SEQ ID NO: 2), indicating that Compound 3205 (SEQ ID NO: 2) inhibits miR-21 and prevents the cells from migrating (FIG. 34B).

Conclusion:

The 8-mer (Compound 3205, SEQ ID NO: 2) targeting miR-21 inhibits the cell migration of PC3 cells compared to untransfected and control transfected cells.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC (#90112714). PC3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Scratch Assay:

150.000 cells were seeded per well in a 6-well plate three days before transfection in order to receive 100% confluency the next day. At 24 hours after transfection, a scratch was made in the cell monolayer with a 200 μl tip. Pictures were taken at 0 h and after 24 hours by using a digital camera coupled to a microscope. The software program Image J was used to determine wound closure.

Example 35 Length Assessment of Fully LNA-Substituted LNA-antimiRs Antagonizing miR-155

We have previously shown a length assessment for miR-21 regarding fully LNA-substituted LNA-antimiRs, and showed that the most potent LNA-antimiRs are 7-, 8- or 9 nt in length. The same experiment was repeated with miR-155. A perfect match target site for miR-155 was cloned into the 3′UTR of the luciferase gene in the reporter plasmid psiCHECK2 and transfected into the mouse RAW macrophage cell line together with fully LNA-substituted LNA-antimiRs of different lengths. Because the endogenous levels of miR-155 are low in the RAW cell line, the cells were treated with 100 ng/ml LPS for 24 hours in order to induce miR-155 accumulation. After 24 hours, luciferase analysis was performed.

Results:

As shown in FIG. 35, the most potent LNA-antimiRs are Compound 3207 (SEQ ID NO: 4) (8 nt) and Compound 3241 (SEQ ID NO: 38) (9 nt), reaching almost a 80% de-repression at only 0.25 nM LNA concentration. The 6-mer (Compound 3244, SEQ ID NO: 978) shows no significant de-repression. Increasing the length to 12-mer to 14-mer (Compound 3242 (SEQ ID NO: 39) and Compound 3243 (SEQ ID NO: 977)) decreased the potency as shown by less efficient de-repression of the miR-155 reporter.

Conclusion:

The most potent fully LNA-substituted LNA-antimiRs targeting miR-155 were an B- and 9-mer (Compound 3207 (SEQ ID NO: 4) and Compound 3241 (SEQ ID NO: 38)).

Materials and Methods:

Cell Line:

The mouse macrophage RAW 264.7 cell line was purchased from ATCC (TIB-71). RAW cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

500.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, RAW 264.7 cells were transfected with 0.3 ug miR-155 perfect match/psiCHECK2 or empty psiCHECK2 vector together with 10 μl Lipofectamine-2000 (Invitrogen) according to the manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. In order to induce miR-155 accumulation, LPS (100 ng/ml) was added to the RAW cells after the 4 hour incubation with the transfection complexes. After another 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and harvested with cell scraper, after which cells were spinned for 5 min at 2.500 rpm. The supernatant was discarded and 50 μl 1× Passive Lysis Buffer (Promega) was added to the cell pellet, after which cells were put on ice for 30 min. The lysed cells were spinned at 10.000 rpm for 30 min after which 20 μl were transferred to a 96-well plate and luciferase measurements were performed according to the manufacturer's instructions (Promega).

Example 36 Plasma Protein Binding for the Fully LNA-Substituted 8-Mer Compound 3205 (SEQ ID NO: 2) Targeting miR-21 (LNA-antimiR-21)

The plasma proteins are not saturated with Compound 3205 (SEQ ID NO:2) at the plasma concentrations in the experiment shown in FIG. 36A. In a wide range of Compound 3205 (SEQ ID NO: 2) concentrations in the plasma the protein binding is around 95% of the Compound 3205 (SEQ ID NO: 2) LNA-antimiR-21 in FIG. 36B. At Compound 3205 (SEQ ID NO: 2) concentrations 50.1 μM (174 μg/mL) the binding capacity of plasma proteins for FAM-labeled Compound 3205 (SEQ ID NO: 2) has not been saturated.

Materials and Methods:

Mouse plasma (100 μL) was spiked with FAM-labeled Compound 3205 (SEQ ID NO: 2) to 0.167, 1.67, 5.01, 10.02, 16.7, 25.05 and 50.1 μM concentrations. The solutions were incubated at 37° C. for 30 minutes. The solutions were transferred to a Microcon Ultracel YM-30 filter (regenerated cellulose 30.000 MWCO). The filters were spun for 20 minutes at 2000 g and at room temperature in a microcentrifuge. The filtrate was diluted 5, 10 and 20 times and 100 μL samples were transferred to a microtiter plate (Polystyrene Black NUNC-237108). The fluorescence was detected using a FLUOstar Optima elisa reader with excitation 458 nm and emission 520 nm. The amount of unbound FAM-labeled Compound 3205 (SEQ ID NO: 2) was calculated from a standard curve derived from filtrated plasma spiked with FAM-labeled Compound 3205 (SEQ ID NO: 2) at 12 different (0.45-1000 nM) concentrations. The numbers were corrected with the recovery number established from filtration experiments with Compound 3205 (SEQ ID NO: 2) concentrations 0.167, 1.67, 5.01, 10.02, 16.7, 25.05 and 50.1 μM in filtrated plasma. The recovery of FAM-labeled Compound 3205 (SEQ ID NO: 2) was 86%.

Example 37 Quantitative Whole Body Autoradiography Study in Female Pigmented Mice after Single Intravenous Administration of ³⁵S-Labelled Compound 3205 (SEQ ID NO: 2) LNA-antimiR-21

In order to determine the biodistribution of a short fully LNA-modified LNA-antimiR (Compound 3205 (SEQ ID NO: 2), 8-mer) a whole body tissue distribution of radioactively labeled compound was done in mice. ³⁵S-labelled Compound 3205 (SEQ ID NO: 2) was dosed to mice with a single intravenous administration and mice were sacrificed at different time-points, ranging from 5 min to 21 days.

TABLE 6(i) Individual tissue concentrations (μg Compound 3205/g tissue) after a single intravenous administration of ³⁵S-labelled Compound 3205 (SEQ ID NO: 2) in female pigmented mice. Max. Conc. of oligo μg Compound Time of max Tissue 3205/g tissue conc. hours T½ hours Adrenal gl. 13.6 0.083 374 Bile 4 1 Bone marrow 7.2 0.083 411 Brain 0.4 0.083 Brown fat 8.8 0.083 Gastric muc. 10.1 0.083 Heart blood 26.2 0.083 10.3 Kidney ctx. 58.7 24 104 Liver 11.8 0.083 588 10.7 24 Lung 13.2 0.083 289 Lymph node 5 0.083 262 2.4 48 Lymph 18.8 4 20.8 168 Myocardium 8.1 0.083 662 Ovary 13 0.083 198 Pancreas 5 0.083 Pituitary gl. 6.7 0.083 Salivary gl. 8.6 0.083 405 5.5 168 skel. Muscle 4.8 0.083 Skin pig. 5.4 0.25 Spleen 9.8 0.083 564 Thymus 3.8 0.083 185 Thyroid gl. 10.9 0.083 592 Urine 328.9 0.083 Uterus 9.6 0.25 177 Uvea of the eye 13.6 0.083 LOQ 0.045 0.083 0.033 24 0.03 168 The figures are mean values of three measurements for each tissue and ratio. The coefficient of variation (CV) is generally about 10%.

TABLE 6(ii) Tissue to liver ratios after single intravenous administration of ³⁵S-labelled Compound 3205 (SEQ ID NO: 2) in female pigmented mice. ³⁵S-Compound 3205 (SEQ ID NO: 2) Animal no 10 11 12 13 14 15 16 17 18 Surv. Time (h) Organ 0.083 0.25 1 h 4 h 24 h 48 h 96 h 168 504 Adrenal gl liver  liver liver  liver liver  liver liver liver liver Bile 1.15 1.08 0.52 0.27 0.24 0.26 0.23 0.18 0.17 Bone marrow 0.03 0.11 0.55 0.10 0.03 0.07 0.04 0.03 0.04 Brain 0.61 0.81 0.55 0.45 0.40 0.48 0.43 0.42 0.34 Brown fat 0.03 0.03 0.01 0.00 0.00 0.00 0.00 0.00 0.00 Gastric muc 0.75 0.57 0.29 0.12 0.07 0.12 0.08 0.10 0.07 Heart blood 0.86 0.71 0.31 0.22 0.10 0.21 0.15 0.16 0.12 Kidney ctx 2.23 1.91 0.74 0.11 0.01 0.00 0.00 0.00 0.00 Liver 2.87 3.94 6.45 6.95 5.51 6.68 3.92 2.24 0.40 Lung 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Lymph node 1.12 0.97 0.63 0.09 0.04 0.04 0.03 0.02 0.02 Lymph 0.43 0.30 0.25 0.19 0.11 0.32 0.20 0.17 0.12 Myocardium 0.82 1.09 1.78 2.78 1.03 2.05 1.62 3.17 1.89 Ovary 0.69 0.63 0.30 0.13 0.10 0.15 0.09 0.11 0.12 Pancreas 1.10 1.40 0.61 0.31 0.27 0.28 0.21 0.21 0.08 Pituitary gland 0.42 0.37 0.22 0.18 0.12 0.17 0.12 0.15 0.11 Salivary gland 0.57 0.54 0.28 0.11 0.15 0.16 0.12 0.10 0.08 Skel. muscle 0.73 0.81 0.38 0.25 0.25 0.42 0.23 0.85 0.24 Skin, pigm. 0.40 0.28 0.14 0.04 0.02 0.04 0.03 0.03 0.03 Spleen 0.34 0.69 0.65 0.36 0.20 0.26 0.20 0.19 0.13 Thymus 0.83 0.86 0.44 0.32 0.24 0.34 0.35 0.29 0.31 Thyroid gland 0.32 0.31 0.14 0.07 0.09 0.08 0.05 0.04 0.02 Urine 0.9 1.2 0.43 0.28 0.25 0.34 0.19 0.26 0.25 Uterus 27.96 39.48 9.90 5.44 0.24 0.39 0.12 0.15 0.03 Uvea of the eye 0.56 1.23 0.65 0.30 0.30 0.07 0.27 0.16 0.08 Conclusions:

Compound 3205 (SEQ ID NO: 2) shows blood clearance of radioactivity with elimination half-lives of 8-10 hours. High levels of radioactivity were registered in the kidney cortex, lymph, liver, bone marrow, spleen, ovary and uterus. The highest level of radioactivity was registered in the kidney cortex showing five times higher levels than that of the liver for Compound 3205 (SEQ ID NO: 2). A strong retention of radioactivity was noticed in the kidney cortex, lymph, liver, bone marrow and spleen for Compound 3205 (SEQ ID NO: 2) LNA-antimiR-21.

Materials and Methods:

Dose Administration:

All mice were weighed before administration. Nine female mice were given 10 mg/kg of ³⁵S-Compound 3205 (SEQ ID NO: 2) intravenously in a tail vein. The volume given to each animal was 10 mL/kg of the test formulation. The specific activity 75.7 μCi/mg. Individual mice were killed 5 min, 15 min, 1 hour, 4 hours, 24 hours, 2 days, 4 days, 7 days and 21 days after administration of Compound 3205 (SEQ ID NO: 2).Whole body autoradiography: The mice were anaesthetized by sevoflurane, and then immediately immersed in heptane, cooled with dry ice to −80° C., ABR-SOP-0130. The frozen carcasses were embedded in a gel of aqueous carboxymethyl cellulose (CMC), frozen in ethanol, cooled with dry ice (−80° C.) and sectioned sagittaly for whole body autoradiography, according to the standard method, ABR-SOP-0131. From each animal 20 μm sections were cut at different levels with a cryomicrotome (Leica CM 3600) at a temperature of about −20° C. The obtained sections were caught on tape (Minnesota Mining and Manufacturing Co., No. 810) and numbered consecutively with radioactive ink. After being freeze-dried at −20° C. for about 24 hours, selected sections were covered with a thin layer of mylar foil, and put on imaging plates (Fuji, Japan). Exposure took place in light tight cassettes in a lead shielding box at −20° C., to protect the image plates from environmental radiation. After exposure the imaging plates were scanned at a pixel size of 50 μm and analyzed by radioluminography using a bioimaging analysis system (Bas 2500, Fuji, Japan), and described in ABR-SOP-0214. A water-soluble standard test solution of ³⁵S radioactivity was mixed with whole blood and used for production of a calibration scale, ABR-SOP-0251. However, the different blood standards were dissolved in 500 uL Soluene-35. 4.5 mL Ultima Gold was then added to the dissolved samples. As ³⁵S and ¹⁴C have very similar energy spectra, a standard ¹⁴C-programme (Packard 2200CA) was used when the radioactivity for the different blood samples was settled.

Pharmacokinetic Calculations:

The ³⁵S radioactivity measured in whole blood and tissues was expressed as nCi/g tissue and recalculated to nmol equiv/g tissue for the pharmacokinetic evaluation. The pharmacokinetic parameters C_(max), t_(1/2) and AUC were determined for the whole blood and tissues by non-compartmental analysis using WinNonlin Professional (Pharsight Corporation, Mountain View, Calif., USA). After intravenous administration, the concentration was extrapolated back to zero and expressed as (C₀). The elimination rate constant λ was estimated by linear regression analysis of the terminal slope of the logarithmic plasma concentration-time curve. The elimination half-life, t_(1/2), was calculated using the equation, t_(1/2)=ln2/λ. The last three time-points above LOQ were used in the elimination half-life calculations, if not stated otherwise.

Example 38 Assessment of Let-7 Inhibition In Vivo by an 8-Mer LNA-antimiR, as Determined Through Ras Protein Quantification in Mouse Lung and Kidney

In order to investigate the possibility to antagonize the abundantly expressed let-7 family in vivo, mice were intravenously (i.v.) injected with an 8-mer LNA-antimiR antagonist or with saline. To measure treatment effect, proteins were isolated from lungs and kidneys. Because the Ras family of proteins (N-Ras, K-Ras, and H-Ras), in particular N-Ras and K-Ras, has previously been shown to be regulated (repressed) by the let-7 family by Johnson et al. (Cell, 2005), the aim was to analyze whether these let-7 targets could be de-repressed in vivo.

Results:

As seen in FIG. 37, the 8-mer LNA-antimiR potently de-repressed Ras protein levels in the kidneys of treated mice, normalized against saline controls. The up-regulation in this organ was more than 3-fold, showing a clear in vivo effect. In the lungs, however, only a minimal (1.2-fold) Ras de-repression was observed (FIG. 1B), suggesting that insufficient amounts of LNA-antimiR has entered this organ in order to inhibit its massive amounts of let-7, as previously described by Johnson et al. (Cancer Research, 2007).

Conclusion:

The 8-mer LNA-antimiR shows a clear effect in regulating target let-7 miRNA in vivo, as evaluated based on Ras protein levels in treated vs. control mice. Whereas the effect seems to be smaller in lungs, Ras levels in the kidney show a substantial up-regulation upon antimiRs-treatment.

Materials and Methods:

Animals and Dosing:

C57BL/6 female mice were treated with 10 mg/kg LNA-antimiR or saline for three consecutive days (0, 1, and 2) and sacrificed on day 4. Tissue samples from lungs and kidneys were snapfrozen and stored at −80° C. until further processing.

Western Blot Analysis:

Lung and kidney proteins from saline and LNA-antimiR-treated mice were separated on NuPAGE Bis Tris 4-12% (Invitrogen), using 100 μg per sample. The proteins were transferred to a nitrocellulose membrane using iBlot (Invitrogen) according to the manufacturer's instructions. Blocking, antibody dilution and detection was performed according to the manufacturer's specifications. For Ras detection, a primary rabbit-anti Ras antibody (SC-3339, Santa Cruz Biotechnology) and a secondary HRP-conjugated swine-anti-rabbit antibody (PO399, Dako) was used, and for tubulin detection, a primary tubulin alpha (MS-581-P1, Neomarkers) and a secondary HRP-conjugated goat-anti-mouse antibody (PO447, Dako) was used.

Example 40 In Vivo Efficacy Assessment of the 8-Mer LNA-antimiR (Compound 3205, SEQ ID NO: 2) in Targeting miR-21, as Determined by Pdcd4 Protein Up-Regulation in Mouse Kidney

We have shown that an 8-mer LNA-antimiR that is fully LNA-modified antagonizes miR-21 and has the ability to regulate the protein levels of the miR-21 target Pdcd4 in vitro. We therefore injected the LNA-antimiR into mice to determine the effects of the LNA-antimiR in vivo. The mice received 25 mg/kg of Compound 3205 (SEQ ID NO: 2) by i.v. injection every other day for 14 days (a total of 5 doses). The mice were sacrificed on day 14, the kidney was removed, and protein was isolated. In order to determine target regulation, Western blot analysis was performed.

Results:

As shown in FIG. 38, treating mice with Compound 3205 (SEQ ID NO: 2)_showed significantly increased Pdcd4 protein levels as compared to the saline control. While the normalized Pdcd4 versus Gapdh ratio was consistent in both saline samples, the protein up-regulation in the two LNA-antimiR-treated with Compound 3205 (SEQ ID NO: 2) mice were measured to 3.3- and 6.3-fold, respectively, demonstrating an in vivo pharmacological effect of the Compound 3205 (SEQ ID NO: 2) 8-mer LNA-antimiR.

Conclusion:

The fully LNA-modified 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) antagonizes miR-21 in vivo, as demonstrated through its ability to de-repress (up-regulate) mouse kidney levels of Pdcd4, a validated miR-21 target.

Materials and Methods:

Animals and Dosing:

C57BL/6 female mice with average of 20 g body weight at first dosing were used in all experiments and received regular chow diet (Altromin no 1324, Brogaarden, Gentofte, Denmark). Substances were formulated in physiological saline (0.9% NaCl). The animals were dozed with LNA-antimiR or saline (0.9% NaCl), receiving an injection of 25 mg/kg every other day for 14 days, a total of 5 doses. Animals were sacrificed on day 14.

Western Blot Analysis:

80 μg kidney tissue from saline or LNA-treated mice was separated on NuPAGE Bis Tris 4-12% (Invitrogen). The proteins were transferred to a nitrocellulose membrane using iBlot (Invitrogen) according to the manufacturer's instructions. The membrane was incubated with Pdcd4 antibody (Bethyl Laboratories), followed by HRP-conjugated swine-anti-rabbit antibody (Dako). As equal loading control, GAPDH (Abcam) was used, followed by HRP-conjugated swine-anti-mouse antibody. The membranes were visualized by chemiluminiscence (ECL, Amersham).

TABLE 1 SEQ ID 9-mer 8-mer 7-mer microRNA MicroRNASequence NO Compound Compound Compound ebv-miR-BART1-3p UAGCACCGCUAUCCACUAUGUC 40 AGCGGTGCT GCGGTGCT CGGTGCT ebv-miR-BART1-5p UCUUAGUGGAAGUGACGUGCUGUG 41 TCCACTAAG CCACTAAG CACTAAG ebv-miR-BART10 UACAUAACCAUGGAGUUGGCUGU 42 TGGTTATGT GGTTATGT GTTATGT ebv-miR-BART10* GCCACCUCUUUGGUUCUGUACA 43 AAGAGGTGG AGAGGTGG GAGGTGG ebv-miR-BART11-3p ACGCACACCAGGCUGACUGCC 44 TGGTGTGCG GGTGTGCG GTGTGCG ebv-miR-BART11-5p UCAGACAGUUUGGUGCGCUAGUUG 45 AACTGTCTG ACTGTCTG CTGTCTG ebv-miR-BART12 UCCUGUGGUGUUUGGUGUGGUU 46 CACCACAGG ACCACAGG CCACAGG ebv-miR-BART13 UGUAACUUGCCAGGGACGGCUGA 47 GCAAGTTAC CAAGTTAC AAGTTAC ebv-miR-BART13* AACCGGCUCGUGGCUCGUACAG 48 CGAGCCGGT GAGCCGGT AGCCGGT ebv-miR-BART14 UAAAUGCUGCAGUAGUAGGGAU 49 GCAGCATTT CAGCATTT AGCATTT ebv-miR-BART14* UACCCUACGCUGCCGAUUUACA 50 GCGTAGGGT CGTAGGGT GTAGGGT ebv-miR-BART15 GUCAGUGGUUUUGUUUCCUUGA 51 AACCACTGA ACCACTGA CCACTGA ebv-miR-BART16 UUAGAUAGAGUGGGUGUGUGCUCU 52 CTCTATCTA TCTATCTA CTATCTA ebv-miR-BART17-3p UGUAUGCCUGGUGUCCCCUUAGU 53 CAGGCATAC AGGCATAC GGCATAC ebv-miR-BART17-5p UAAGAGGACGCAGGCAUACAAG 54 CGTCCTCTT GTCCTCTT TCCTCTT ebv-miR-BART18-3p UAUCGGAAGUUUGGGCUUCGUC 55 ACTTCCGAT CTTCCGAT TTCCGAT ebv-miR-BART18-5p UCAAGUUCGCACUUCCUAUACA 56 GCGAACTTG CGAACTTG GAACTTG ebv-miR-BART19-3p UUUUGUUUGCUUGGGAAUGCU 57 GCAAACAAA CAAACAAA AAACAAA ebv-miR-BART19-5p ACAUUCCCCGCAAACAUGACAUG 58 CGGGGAATG GGGGAATG GGGAATG ebv-miR-BART2-3p AAGGAGCGAUUUGGAGAAAAUAAA 59 ATCGCTCCT TCGCTCCT CGCTCCT ebv-miR-BART2-5p UAUUUUCUGCAUUCGCCCUUGC 60 GCAGAAAAT CAGAAAAT AGAAAAT ebv-miR-BART20-3p CAUGAAGGCACAGCCUGUUACC 61 TGCCTTCAT GCCTTCAT CCTTCAT ebv-miR-BART20-5p UAGCAGGCAUGUCUUCAUUCC 62 ATGCCTGCT TGCCTGCT GCCTGCT ebv-miR-BART3 CGCACCACUAGUCACCAGGUGU 63 TAGTGGTGC AGTGGTGC GTGGTGC ebv-miR-BART3* ACCUAGUGUUAGUGUUGUGCU 64 AACACTAGG ACACTAGG CACTAGG ebv-miR-BART4 GACCUGAUGCUGCUGGUGUGCU 65 GCATCAGGT CATCAGGT ATCAGGT ebv-miR-BART5 CAAGGUGAAUAUAGCUGCCCAUCG 66 ATTCACCTT TTCACCTT TCACCTT ebv-miR-BART6-3p CGGGGAUCGGACUAGCCUUAGA 67 CCGATCCCC CGATCCCC GATCCCC ebv-miR-BART6-5p UAAGGUUGGUCCAAUCCAUAGG 68 ACCAACCTT CCAACCTT CAACCTT ebv-miR-BART7 CAUCAUAGUCCAGUGUCCAGGG 69 GACTATGAT ACTATGAT CTATGAT ebv-miR-BART7* CCUGGACCUUGACUAUGAAACA 70 AAGGTCCAG AGGTCCAG GGTCCAG ebv-miR-BART8 UACGGUUUCCUAGAUUGUACAG 71 GGAAACCGT GAAACCGT AAACCGT ebv-miR-BART8* GUCACAAUCUAUGGGGUCGUAGA 72 AGATTGTGA GATTGTGA ATTGTGA ebv-miR-BART9 UAACACUUCAUGGGUCCCGUAGU 73 TGAAGTGTT GAAGTGTT AAGTGTT ebv-miR-BART9* UACUGGACCCUGAAUUGGAAAC 74 GGGTCCAGT GGTCCAGT GTCCAGT ebv-miR-BHRF1-1 UAACCUGAUCAGCCCCGGAGUU 75 GATCAGGTT ATCAGGTT TCAGGTT ebv-miR-BHRF1-2 UAUCUUUUGCGGCAGAAAUUGA 76 GCAAAAGAT CAAAAGAT AAAAGAT ebv-miR-BHRF1-2* AAAUUCUGUUGCAGCAGAUAGC 77 AACAGAATT ACAGAATT CAGAATT ebv-miR-BHRF1-3 UAACGGGAAGUGUGUAAGCACA 78 CTTCCCGTT TTCCCGTT TCCCGTT hcmv-miR-UL112 AAGUGACGGUGAGAUCCAGGCU 79 ACCGTCACT CCGTCACT CGTCACT hcmv-miR-UL148D UCGUCCUCCCCUUCUUCACCG 80 GGGAGGACG GGAGGACG GAGGACG hcmv-miR-UL22A UAACUAGCCUUCCCGUGAGA 81 AGGCTAGTT GGCTAGTT GCTAGTT hcmv-miR-UL22A* UCACCAGAAUGCUAGUUUGUAG 82 ATTCTGGTG TTCTGGTG TCTGGTG hcmv-miR-UL36 UCGUUGAAGACACCUGGAAAGA 83 TCTTCAACG CTTCAACG TTCAACG hcmv-miR-UL36* UUUCCAGGUGUUUUCAACGUGC 84 CACCTGGAA ACCTGGAA CCTGGAA hcmv-miR-UL70-3p GGGGAUGGGCUGGCGCGCGG 85 GCCCATCCC CCCATCCC CCATCCC hcmv-miR-UL70-5p UGCGUCUCGGCCUCGUCCAGA 86 CCGAGACGC CGAGACGC GAGACGC hcmv-miR-US25-1 AACCGCUCAGUGGCUCGGACC 87 CTGAGCGGT TGAGCGGT GAGCGGT hcmv-miR-US25-1* UCCGAACGCUAGGUCGGUUCUC 88 AGCGTTCGG GCGTTCGG CGTTCGG hcmv-miR-US25-2- AUCCACUUGGAGAGCUCCCGCGG 89 CCAAGTGGA CAAGTGGA AAGTGGA 3p hcmv-miR-US25-2- AGCGGUCUGUUCAGGUGGAUGA 90 ACAGACCGC CAGACCGC AGACCGC 5p hcmv-miR-US33-3p UCACGGUCCGAGCACAUCCA 91 CGGACCGTG GGACCGTG GACCGTG hcmv-miR-US33-5p GAUUGUGCCCGGACCGUGGGCG 92 GGGCACAAT GGCACAAT GCACAAT hcmv-miR-US4 CGACAUGGACGUGCAGGGGGAU 93 GTCCATGTC TCCATGTC CCATGTC hcmv-miR-US5-1 UGACAAGCCUGACGAGAGCGU 94 AGGCTTGTC GGCTTGTC GCTTGTC hcmv-miR-US5-2 UUAUGAUAGGUGUGACGAUGUC 95 CCTATCATA CTATCATA TATCATA hsa-let-7a UGAGGUAGUAGGUUGUAUAGUU 96 TACTACCTC (SEQ ID NO:31) ACTACCTC (SEQ ID NO:25) CTACCTC (SEQ ID NO:30) hsa-let-7a* CUAUACAAUCUACUGUCUUUC 97 GATTGTATA ATTGTATA TTGTATA hsa-let-7b UGAGGUAGUAGGUUGUGUGGUU 98 TACTACCTC (SEQ ID NO:1002) ACTACCTC (SEQ ID NO:1003) CTACCTC (SEQ ID NO:1004) hsa-let-7b* CUAUACAACCUACUGCCUUCCC 99 GGTTGTATA GTTGTATA TTGTATA hsa-let-7c UGAGGUAGUAGGUUGUAUGGUU 100 TACTACCTC (SEQ ID NO:1006) ACTACCTC (SEQ ID NO:1007) CTACCTC (SEQ ID NO:1008) hsa-let-7c* UAGAGUUACACCCUGGGAGUUA 101 TGTAACTCT GTAACTCT TAACTCT hsa-let-7d AGAGGUAGUAGGUUGCAUAGUU 102 TACTACCTC (SEQ ID NO:1010) ACTACCTC (SEQ ID NO:1011) CTACCTC (SEQ ID NO:1012) hsa-let-7d* CUAUACGACCUGCUGCCUUUCU 103 GGTCGTATA GTCGTATA TCGTATA hsa-let-7e UGAGGUAGGAGGUUGUAUAGUU 104 TCCTACCTC (SEQ ID NO:1014) CCTACCTC (SEQ ID NO:1015) CTACCTC (SEQ ID NO:1016) hsa-let-7e* CUAUACGGCCUCCUAGCUUUCC 105 GGCCGTATA GCCGTATA CCGTATA hsa-let-7f UGAGGUAGUAGAUUGUAUAGUU 106 TACTACCTC (SEQ ID NO:1018) ACTACCTC (SEQ ID NO:1019) CTACCTC (SEQ ID NO:1020) hsa-let-7f-1* CUAUACAAUCUAUUGCCUUCCC 107 GATTGTATA ATTGTATA TTGTATA hsa-let-7f-2* CUAUACAGUCUACUGUCUUUCC 108 GACTGTATA ACTGTATA CTGTATA hsa-let-7g UGAGGUAGUAGUUUGUACAGUU 109 TACTACCTC (SEQ ID NO:1022) ACTACCTC (SEQ ID NO:1023) CTACCTC (SEQ ID NO:1024) hsa-let-7g* CUGUACAGGCCACUGCCUUGC 110 GCCTGTACA CCTGTACA CTGTACA hsa-let-7i UGAGGUAGUAGUUUGUGCUGUU 111 TACTACCTC (SEQ ID NO:1026) ACTACCTC (SEQ ID NO:1027) CTACCTC (SEQ ID NO:1028) hsa-let-7i* CUGCGCAAGCUACUGCCUUGCU 112 GCTTGCGCA CTTGCGCA TTGCGCA hsa-miR-1 UGGAAUGUAAAGAAGUAUGUAU 113 TTACATTCC TACATTCC ACATTCC hsa-miR-100 AACCCGUAGAUCCGAACUUGUG 114 TCTACGGGT CTACGGGT TACGGGT hsa-miR-100* CAAGCUUGUAUCUAUAGGUAUG 115 TACAAGCTT ACAAGCTT CAAGCTT hsa-miR-101 UACAGUACUGUGAUAACUGAA 116 CAGTACTGT AGTACTGT GTACTGT hsa-miR-101* CAGUUAUCACAGUGCUGAUGCU 117 GTGATAACT TGATAACT GATAACT hsa-miR-103 AGCAGCAUUGUACAGGGCUAUGA 118 CAATGCTGC AATGCTGC ATGCTGC hsa-miR-103-as UCAUAGCCCUGUACAAUGCUGCU 119 AGGGCTATG GGGCTATG GGCTATG hsa-miR-105 UCAAAUGCUCAGACUCCUGUGGU 120 GAGCATTTG AGCATTTG GCATTTG hsa-miR-105* ACGGAUGUUUGAGCAUGUGCUA 121 AAACATCCG AACATCCG ACATCCG hsa-miR-106a AAAAGUGCUUACAGUGCAGGUAG 122 AAGCACTTT AGCACTTT GCACTTT hsa-miR-106a* CUGCAAUGUAAGCACUUCUUAC 123 TACATTGCA ACATTGCA CATTGCA hsa-miR-106b UAAAGUGCUGACAGUGCAGAU 124 CAGCACTTT (SEQ ID NO:989) AGCACTTT (SEQ ID NO:19) GCACTTT (SEQ ID NO:990) hsa-miR-106b* CCGCACUGUGGGUACUUGCUGC 125 CACAGTGCG ACAGTGCG CAGTGCG hsa-miR-107 AGCAGCAUUGUACAGGGCUAUCA 126 CAATGCTGC AATGCTGC ATGCTGC hsa-miR-10a UACCCUGUAGAUCCGAAUUUGUG 127 CTACAGGGT TACAGGGT ACAGGGT hsa-miR-10a* CAAAUUCGUAUCUAGGGGAAUA 128 TACGAATTT ACGAATTT CGAATTT hsa-miR-10b UACCCUGUAGAACCGAAUUUGUG 129 CTACAGGGT TACAGGGT ACAGGGT hsa-miR-10b* ACAGAUUCGAUUCUAGGGGAAU 130 TCGAATCTG CGAATCTG GAATCTG hsa-miR-1178 UUGCUCACUGUUCUUCCCUAG 131 CAGTGAGCA AGTGAGCA GTGAGCA hsa-miR-1179 AAGCAUUCUUUCAUUGGUUGG 132 AAGAATGCT AGAATGCT GAATGCT hsa-miR-1180 UUUCCGGCUCGCGUGGGUGUGU 133 GAGCCGGAA AGCCGGAA GCCGGAA hsa-miR-1181 CCGUCGCCGCCACCCGAGCCG 134 GCGGCGACG CGGCGACG GGCGACG hsa-miR-1182 GAGGGUCUUGGGAGGGAUGUGAC 135 CAAGACCCT AAGACCCT AGACCCT hsa-miR-1183 CACUGUAGGUGAUGGUGAGAGUGGGCA 136 ACCTACAGT CCTACAGT CTACAGT hsa-miR-1184 CCUGCAGCGACUUGAUGGCUUCC 137 TCGCTGCAG CGCTGCAG GCTGCAG hsa-miR-1185 AGAGGAUACCCUUUGUAUGUU 138 GGTATCCTC GTATCCTC TATCCTC hsa-miR-1197 UAGGACACAUGGUCUACUUCU 139 ATGTGTCCT TGTGTCCT GTGTCCT hsa-miR-1200 CUCCUGAGCCAUUCUGAGCCUC 140 GGCTCAGGA GCTCAGGA CTCAGGA hsa-miR-1201 AGCCUGAUUAAACACAUGCUCUGA 141 TAATCAGGC AATCAGGC ATCAGGC hsa-miR-1202 GUGCCAGCUGCAGUGGGGGAG 142 CAGCTGGCA AGCTGGCA GCTGGCA hsa-miR-1203 CCCGGAGCCAGGAUGCAGCUC 143 TGGCTCCGG GGCTCCGG GCTCCGG hsa-miR-1204 UCGUGGCCUGGUCUCCAUUAU 144 CAGGCCACG AGGCCACG GGCCACG hsa-miR-1205 UCUGCAGGGUUUGCUUUGAG 145 ACCCTGCAG CCCTGCAG CCTGCAG hsa-miR-1206 UGUUCAUGUAGAUGUUUAAGC 146 TACATGAAC ACATGAAC CATGAAC hsa-miR-1207-3p UCAGCUGGCCCUCAUUUC 147 GGCCAGCTG GCCAGCTG CCAGCTG hsa-miR-1207-5p UGGCAGGGAGGCUGGGAGGGG 148 CTCCCTGCC TCCCTGCC CCCTGCC hsa-miR-1208 UCACUGUUCAGACAGGCGGA 149 TGAACAGTG GAACAGTG AACAGTG hsa-miR-122 UGGAGUGUGACAAUGGUGUUUG 150 TCACACTCC (SEQ ID NO:982) CACACTCC (SEQ ID NO:983) ACACTCC (SEQ ID NO:984) hsa-miR-122* AACGCCAUUAUCACACUAAAUA 151 TAATGGCGT AATGGCGT ATGGCGT hsa-miR-1224-3p CCCCACCUCCUCUCUCCUCAG 152 GGAGGTGGG GAGGTGGG AGGTGGG hsa-miR-1224-5p GUGAGGACUCGGGAGGUGG 153 GAGTCCTCA AGTCCTCA GTCCTCA hsa-miR-1225-3p UGAGCCCCUGUGCCGCCCCCAG 154 CAGGGGCTC AGGGGCTC GGGGCTC hsa-miR-1225-5p GUGGGUACGGCCCAGUGGGGGG 155 CCGTACCCA CGTACCCA GTACCCA hsa-miR-1226 UCACCAGCCCUGUGUUCCCUAG 156 GGGCTGGTG GGCTGGTG GCTGGTG hsa-miR-1226* GUGAGGGCAUGCAGGCCUGGAUGGGG 157 ATGCCCTCA TGCCCTCA GCCCTCA hsa-miR-1227 CGUGCCACCCUUUUCCCCAG 158 GGGTGGCAC GGTGGCAC GTGGCAC hsa-miR-1228 UCACACCUGCCUCGCCCCCC 159 GCAGGTGTG CAGGTGTG AGGTGTG hsa-miR-1228* GUGGGCGGGGGCAGGUGUGUG 160 CCCCGCCCA CCCGCCCA CCGCCCA hsa-miR-1229 CUCUCACCACUGCCCUCCCACAG 161 GTGGTGAGA TGGTGAGA GGTGAGA hsa-miR-1231 GUGUCUGGGCGGACAGCUGC 162 GCCCAGACA CCCAGACA CCAGACA hsa-miR-1233 UGAGCCCUGUCCUCCCGCAG 163 ACAGGGCTC CAGGGCTC AGGGCTC hsa-miR-1234 UCGGCCUGACCACCCACCCCAC 164 GTCAGGCCG TCAGGCCG CAGGCCG hsa-miR-1236 CCUCUUCCCCUUGUCUCUCCAG 165 GGGGAAGAG GGGAAGAG GGAAGAG hsa-miR-1237 UCCUUCUGCUCCGUCCCCCAG 166 AGCAGAAGG GCAGAAGG CAGAAGG hsa-miR-1238 CUUCCUCGUCUGUCUGCCCC 167 GACGAGGAA ACGAGGAA CGAGGAA hsa-miR-124 UAAGGCACGCGGUGAAUGCC 168 GCGTGCCTT CGTGCCTT GTGCCTT hsa-miR-124* CGUGUUCACAGCGGACCUUGAU 169 TGTGAACAC GTGAACAC TGAACAC hsa-miR-1243 AACUGGAUCAAUUAUAGGAGUG 170 TGATCCAGT GATCCAGT ATCCAGT hsa-miR-1244 AAGUAGUUGGUUUGUAUGAGAUGGUU 171 CCAACTACT CAACTACT AACTACT hsa-miR-1245 AAGUGAUCUAAAGGCCUACAU 172 TAGATCACT AGATCACT GATCACT hsa-miR-1246 AAUGGAUUUUUGGAGCAGG 173 AAAATCCAT AAATCCAT AATCCAT hsa-miR-1247 ACCCGUCCCGUUCGUCCCCGGA 174 CGGGACGGG GGGACGGG GGACGGG hsa-miR-1248 ACCUUCUUGUAUAAGCACUGUGCUAAA 175 ACAAGAAGG CAAGAAGG AAGAAGG hsa-miR-1249 ACGCCCUUCCCCCCCUUCUUCA 176 GGAAGGGCG GAAGGGCG AAGGGCG hsa-miR-1250 ACGGUGCUGGAUGUGGCCUUU 177 CCAGCACCG CAGCACCG AGCACCG hsa-miR-1251 ACUCUAGCUGCCAAAGGCGCU 178 CAGCTAGAG AGCTAGAG GCTAGAG hsa-miR-1252 AGAAGGAAAUUGAAUUCAUUUA 179 ATTTCCTTC TTTCCTTC TTCCTTC hsa-miR-1253 AGAGAAGAAGAUCAGCCUGCA 180 CTTCTTCTC TTCTTCTC TCTTCTC hsa-miR-1254 AGCCUGGAAGCUGGAGCCUGCAGU 181 CTTCCAGGC TTCCAGGC TCCAGGC hsa-miR-1255a AGGAUGAGCAAAGAAAGUAGAUU 182 TGCTCATCC GCTCATCC CTCATCC hsa-miR-1255b CGGAUGAGCAAAGAAAGUGGUU 183 TGCTCATCC GCTCATCC CTCATCC hsa-miR-1256 AGGCAUUGACUUCUCACUAGCU 184 GTCAATGCC TCAATGCC CAATGCC hsa-miR-1257 AGUGAAUGAUGGGUUCUGACC 185 ATCATTCAC TCATTCAC CATTCAC hsa-miR-1258 AGUUAGGAUUAGGUCGUGGAA 186 AATCCTAAC ATCCTAAC TCCTAAC hsa-miR-1259 AUAUAUGAUGACUUAGCUUUU 187 CATCATATA ATCATATA TCATATA hsa-miR-125a-3p ACAGGUGAGGUUCUUGGGAGCC 188 CCTCACCTG CTCACCTG TCACCTG hsa-miR-125a-5p UCCCUGAGACCCUUUAACCUGUGA 189 GTCTCAGGG TCTCAGGG CTCAGGG hsa-miR-125b UCCCUGAGACCCUAACUUGUGA 190 GTCTCAGGG TCTCAGGG CTCAGGG hsa-miR-125b-1* ACGGGUUAGGCUCUUGGGAGCU 191 CCTAACCCG CTAACCCG TAACCCG hsa-miR-125b-2* UCACAAGUCAGGCUCUUGGGAC 192 TGACTTGTG GACTTGTG ACTTGTG hsa-miR-126 UCGUACCGUGAGUAAUAAUGCG 193 CACGGTACG ACGGTACG CGGTACG hsa-miR-126* CAUUAUUACUUUUGGUACGCG 194 AGTAATAAT GTAATAAT TAATAAT hsa-miR-1260 AUCCCACCUCUGCCACCA 195 GAGGTGGGA AGGTGGGA GGTGGGA hsa-miR-1261 AUGGAUAAGGCUUUGGCUU 196 CCTTATCCA CTTATCCA TTATCCA hsa-miR-1262 AUGGGUGAAUUUGUAGAAGGAU 197 ATTCACCCA TTCACCCA TCACCCA hsa-miR-1263 AUGGUACCCUGGCAUACUGAGU 198 AGGGTACCA GGGTACCA GGTACCA hsa-miR-1264 CAAGUCUUAUUUGAGCACCUGUU 199 ATAAGACTT TAAGACTT AAGACTT hsa-miR-1265 CAGGAUGUGGUCAAGUGUUGUU 200 CCACATCCT CACATCCT ACATCCT hsa-miR-1266 CCUCAGGGCUGUAGAACAGGGCU 201 AGCCCTGAG GCCCTGAG CCCTGAG hsa-miR-1267 CCUGUUGAAGUGUAAUCCCCA 202 CTTCAACAG TTCAACAG TCAACAG hsa-miR-1268 CGGGCGUGGUGGUGGGGG 203 ACCACGCCC CCACGCCC CACGCCC hsa-miR-1269 CUGGACUGAGCCGUGCUACUGG 204 CTCAGTCCA TCAGTCCA CAGTCCA hsa-miR-127-3p UCGGAUCCGUCUGAGCUUGGCU 205 ACGGATCCG CGGATCCG GGATCCG hsa-miR-127-5p CUGAAGCUCAGAGGGCUCUGAU 206 TGAGCTTCA GAGCTTCA AGCTTCA hsa-miR-1270 CUGGAGAUAUGGAAGAGCUGUGU 207 ATATCTCCA TATCTCCA ATCTCCA hsa-miR-1271 CUUGGCACCUAGCAAGCACUCA 208 AGGTGCCAA GGTGCCAA GTGCCAA hsa-miR-1272 GAUGAUGAUGGCAGCAAAUUCUGAAA 209 CATCATCAT ATCATCAT TCATCAT hsa-miR-1273 GGGCGACAAAGCAAGACUCUUUCUU 210 TTTGTCGCC TTGTCGCC TGTCGCC hsa-miR-1274a GUCCCUGUUCAGGCGCCA 211 GAACAGGGA AACAGGGA ACAGGGA hsa-miR-1274b UCCCUGUUCGGGCGCCA 212 CGAACAGGG GAACAGGG AACAGGG hsa-miR-1275 GUGGGGGAGAGGCUGUC 213 TCTCCCCCA CTCCCCCA TCCCCCA hsa-miR-1276 UAAAGAGCCCUGUGGAGACA 214 GGGCTCTTT GGCTCTTT GCTCTTT hsa-miR-1277 UACGUAGAUAUAUAUGUAUUUU 215 TATCTACGT ATCTACGT TCTACGT hsa-miR-1278 UAGUACUGUGCAUAUCAUCUAU 216 CACAGTACT ACAGTACT CAGTACT hsa-miR-1279 UCAUAUUGCUUCUUUCU 217 AGCAATATG GCAATATG CAATATG hsa-miR-128 UCACAGUGAACCGGUCUCUUU 218 TTCACTGTG TCACTGTG CACTGTG hsa-miR-1280 UCCCACCGCUGCCACCC 219 AGCGGTGGG GCGGTGGG CGGTGGG hsa-miR-1281 UCGCCUCCUCCUCUCCC 220 GAGGAGGCG AGGAGGCG GGAGGCG hsa-miR-1282 UCGUUUGCCUUUUUCUGCUU 221 AGGCAAACG GGCAAACG GCAAACG hsa-miR-1283 UCUACAAAGGAAAGCGCUUUCU 222 CCTTTGTAG CTTTGTAG TTTGTAG hsa-miR-1284 UCUAUACAGACCCUGGCUUUUC 223 TCTGTATAG CTGTATAG TGTATAG hsa-miR-1285 UCUGGGCAACAAAGUGAGACCU 224 GTTGCCCAG TTGCCCAG TGCCCAG hsa-miR-1286 UGCAGGACCAAGAUGAGCCCU 225 TGGTCCTGC GGTCCTGC GTCCTGC hsa-miR-1287 UGCUGGAUCAGUGGUUCGAGUC 226 TGATCCAGC GATCCAGC ATCCAGC hsa-miR-1288 UGGACUGCCCUGAUCUGGAGA 227 GGGCAGTCC GGCAGTCC GCAGTCC hsa-miR-1289 UGGAGUCCAGGAAUCUGCAUUUU 228 CTGGACTCC TGGACTCC GGACTCC hsa-miR-129* AAGCCCUUACCCCAAAAAGUAU 229 GTAAGGGCT TAAGGGCT AAGGGCT hsa-miR-129-3p AAGCCCUUACCCCAAAAAGCAU 230 GTAAGGGCT TAAGGGCT AAGGGCT hsa-miR-129-5p CUUUUUGCGGUCUGGGCUUGC 231 CCGCAAAAA CGCAAAAA GCAAAAA hsa-miR-1290 UGGAUUUUUGGAUCAGGGA 232 CAAAAATCC AAAAATCC AAAATCC hsa-miR-1291 UGGCCCUGACUGAAGACCAGCAGU 233 GTCAGGGCC TCAGGGCC CAGGGCC hsa-miR-1292 UGGGAACGGGUUCCGGCAGACGCUG 234 CCCGTTCCC CCGTTCCC CGTTCCC hsa-miR-1293 UGGGUGGUCUGGAGAUUUGUGC 235 AGACCACCC GACCACCC ACCACCC hsa-miR-1294 UGUGAGGUUGGCAUUGUUGUCU 236 CAACCTCAC AACCTCAC ACCTCAC hsa-miR-1295 UUAGGCCGCAGAUCUGGGUGA 237 TGCGGCCTA GCGGCCTA CGGCCTA hsa-miR-1296 UUAGGGCCCUGGCUCCAUCUCC 238 AGGGCCCTA GGGCCCTA GGCCCTA hsa-miR-1297 UUCAAGUAAUUCAGGUG 239 ATTACTTGA TTACTTGA TACTTGA hsa-miR-1298 UUCAUUCGGCUGUCCAGAUGUA 240 GCCGAATGA CCGAATGA CGAATGA hsa-miR-1299 UUCUGGAAUUCUGUGUGAGGGA 241 AATTCCAGA ATTCCAGA TTCCAGA hsa-miR-1300 UUGAGAAGGAGGCUGCUG 242 TCCTTCTCA CCTTCTCA CTTCTCA hsa-miR-1301 UUGCAGCUGCCUGGGAGUGACUUC 243 GCAGCTGCA CAGCTGCA AGCTGCA hsa-miR-1302 UUGGGACAUACUUAUGCUAAA 244 TATGTCCCA ATGTCCCA TGTCCCA hsa-miR-1303 UUUAGAGACGGGGUCUUGCUCU 245 CGTCTCTAA GTCTCTAA TCTCTAA hsa-miR-1304 UUUGAGGCUACAGUGAGAUGUG 246 TAGCCTCAA AGCCTCAA GCCTCAA hsa-miR-1305 UUUUCAACUCUAAUGGGAGAGA 247 GAGTTGAAA AGTTGAAA GTTGAAA hsa-miR-1306 ACGUUGGCUCUGGUGGUG 248 GAGCCAACG AGCCAACG GCCAACG hsa-miR-1307 ACUCGGCGUGGCGUCGGUCGUG 249 CACGCCGAG ACGCCGAG CGCCGAG hsa-miR-1308 GCAUGGGUGGUUCAGUGG 250 CCACCCATG CACCCATG ACCCATG hsa-miR-130a CAGUGCAAUGUUAAAAGGGCAU 251 CATTGCACT ATTGCACT TTGCACT hsa-miR-130a* UUCACAUUGUGCUACUGUCUGC 252 ACAATGTGA CAATGTGA AATGTGA hsa-miR-130b CAGUGCAAUGAUGAAAGGGCAU 253 CATTGCACT ATTGCACT TTGCACT hsa-miR-130b* ACUCUUUCCCUGUUGCACUAC 254 GGGAAAGAG GGAAAGAG GAAATAG hsa-miR-132 UAACAGUCUACAGCCAUGGUCG 255 TAGACTGTT AGACTGTT GACTGTT hsa-miR-132* ACCGUGGCUUUCGAUUGUUACU 256 AAGCCACGG AGCCACGG GCCACGG hsa-miR-1321 CAGGGAGGUGAAUGUGAU 257 CACCTCCCT ACCTCCCT CCTCCCT hsa-miR-1322 GAUGAUGCUGCUGAUGCUG 258 CAGCATCAT AGCATCAT GCATCAT hsa-miR-1323 UCAAAACUGAGGGGCAUUUUCU 259 TCAGTTTTG CAGTTTTG AGTTTTG hsa-miR-1324 CCAGACAGAAUUCUAUGCACUUUC 260 TTCTGTCTG TCTGTCTG CTGTCTG hsa-miR-133a UUUGGUCCCCUUCAACCAGCUG 261 GGGGACCAA GGGACCAA GGACCAA hsa-miR-133b UUUGGUCCCCUUCAACCAGCUA 262 GGGGACCAA GGGACCAA GGACCAA hsa-miR-134 UGUGACUGGUUGACCAGAGGGG 263 ACCAGTCAC CCAGTCAC CAGTCAC hsa-miR-135a UAUGGCUUUUUAUUCCUAUGUGA 264 AAAAGCCAT AAAGCCAT AAGCCAT hsa-miR-135a* UAUAGGGAUUGGAGCCGUGGCG 265 AATCCCTAT ATCCCTAT TCCCTAT hsa-miR-135b UAUGGCUUUUCAUUCCUAUGUGA 266 AAAAGCCAT AAAGCCAT AAGCCAT hsa-miR-135b* AUGUAGGGCUAAAAGCCAUGGG 267 AGCCCTACA GCCCTACA CCCTACA hsa-miR-136 ACUCCAUUUGUUUUGAUGAUGGA 268 CAAATGGAG AAATGGAG AATGGAG hsa-miR-136* CAUCAUCGUCUCAAAUGAGUCU 269 GACGATGAT ACGATGAT CGATGAT hsa-miR-137 UUAUUGCUUAAGAAUACGCGUAG 270 TAAGCAATA AAGCAATA AGCAATA hsa-miR-138 AGCUGGUGUUGUGAAUCAGGCCG 271 AACACCAGC ACACCAGC CACCAGC hsa-miR-138-1* GCUACUUCACAACACCAGGGCC 272 GTGAAGTAG TGAAGTAG GAAGTAG hsa-miR-138-2* GCUAUUUCACGACACCAGGGUU 273 GTGAAATAG TGAAATAG GAAATAG hsa-miR-139-3p GGAGACGCGGCCCUGUUGGAGU 274 CCGCGTCTC CGCGTCTC GCGTCTC hsa-miR-139-5p UCUACAGUGCACGUGUCUCCAG 275 GCACTGTAG CACTGTAG ACTGTAG hsa-miR-140-3p UACCACAGGGUAGAACCACGG 276 CCCTGTGGT CCTGTGGT CTGTGGT hsa-miR-140-5p CAGUGGUUUUACCCUAUGGUAG 277 AAAACCACT AAACCACT AACCACT hsa-miR-141 UAACACUGUCUGGUAAAGAUGG 278 GACAGTGTT ACAGTGTT CAGTGTT hsa-miR-141* CAUCUUCCAGUACAGUGUUGGA 279 CTGGAAGAT TGGAAGAT GGAAGAT hsa-miR-142-3p UGUAGUGUUUCCUACUUUAUGGA 280 AAACACTAC AACACTAC ACACTAC hsa-miR-142-5p CAUAAAGUAGAAAGCACUACU 281 CTACTTTAT TACTTTAT ACTTTAT hsa-miR-143 UGAGAUGAAGCACUGUAGCUC 282 CTTCATCTC TTCATCTC TCATCTC hsa-miR-143* GGUGCAGUGCUGCAUCUCUGGU 283 GCACTGCAC CACTGCAC ACTGCAC hsa-miR-144 UACAGUAUAGAUGAUGUACU 284 CTATACTGT TATACTGT ATACTGT hsa-miR-144* GGAUAUCAUCAUAUACUGUAAG 285 GATGATATC ATGATATC TGATATC hsa-miR-145 GUCCAGUUUUCCCAGGAAUCCCU 286 AAAACTGGA AAACTGGA AACTGGA hsa-miR-145* GGAUUCCUGGAAAUACUGUUCU 287 CCAGGAATC CAGGAATC AGGAATC hsa-miR-1468 CUCCGUUUGCCUGUUUCGCUG 288 GCAAACGGA CAAACGGA AAACGGA hsa-miR-1469 CUCGGCGCGGGGCGCGGGCUCC 289 CCGCGCCGA CGCGCCGA GCGCCGA hsa-miR-146a UGAGAACUGAAUUCCAUGGGUU 290 TCAGTTCTC CAGTTCTC AGTTCTC hsa-miR-146a* CCUCUGAAAUUCAGUUCUUCAG 291 ATTTCAGAG TTTCAGAG TTCAGAG hsa-miR-146b-3p UGCCCUGUGGACUCAGUUCUGG 292 CCACAGGGC CACAGGGC ACAGGGC hsa-miR-146b-5p UGAGAACUGAAUUCCAUAGGCU 293 TCAGTTCTC CAGTTCTC AGTTCTC hsa-miR-147 GUGUGUGGAAAUGCUUCUGC 294 TTCCACACA TCCACACA CCACACA hsa-miR-1470 GCCCUCCGCCCGUGCACCCCG 295 GGCGGAGGG GCGGAGGG CGGAGGG hsa-miR-1471 GCCCGCGUGUGGAGCCAGGUGU 296 ACACGCGGG CACGCGGG ACGCGGG hsa-miR-147b GUGUGCGGAAAUGCUUCUGCUA 297 TTCCGCACA TCCGCACA CCGCACA hsa-miR-148a UCAGUGCACUACAGAACUUUGU 298 AGTGCACTG GTGCACTG TGCACTG hsa-miR-148a* AAAGUUCUGAGACACUCCGACU 299 TCAGAACTT CAGAACTT AGAACTT hsa-miR-148b UCAGUGCAUCACAGAACUUUGU 300 GATGCACTG ATGCACTG TGCACTG hsa-miR-148b* AAGUUCUGUUAUACACUCAGGC 301 AACAGAACT ACAGAACT CAGAACT hsa-miR-149 UCUGGCUCCGUGUCUUCACUCCC 302 CGGAGCCAG GGAGCCAG GAGCCAG hsa-miR-149* AGGGAGGGACGGGGGCUGUGC 303 GTCCCTCCC TCCCTCCC CCCTCCC hsa-miR-150 UCUCCCAACCCUUGUACCAGUG 304 GGTTGGGAG GTTGGGAG TTGGGAG hsa-miR-150* CUGGUACAGGCCUGGGGGACAG 305 CCTGTACCA CTGTACCA TGTACCA hsa-miR-151-3p CUAGACUGAAGCUCCUUGAGG 306 TTCAGTCTA TCAGTCTA CAGTCTA hsa-miR-151-5p UCGAGGAGCUCACAGUCUAGU 307 AGCTCCTCG GCTCCTCG CTCCTCG hsa-miR-152 UCAGUGCAUGACAGAACUUGG 308 CATGCACTG ATGCACTG TGCACTG hsa-miR-153 UUGCAUAGUCACAAAAGUGAUC 309 GACTATGCA ACTATGCA CTATGCA hsa-miR-1537 AAAACCGUCUAGUUACAGUUGU 310 AGACGGTTT GACGGTTT ACGGTTT hsa-miR-1538 CGGCCCGGGCUGCUGCUGUUCCU 311 GCCCGGGCC CCCGGGCC CCGGGCC hsa-miR-1539 UCCUGCGCGUCCCAGAUGCCC 312 ACGCGCAGG CGCGCAGG GCGCAGG hsa-miR-154 UAGGUUAUCCGUGUUGCCUUCG 313 GGATAACCT GATAACCT ATAACCT hsa-miR-154* AAUCAUACACGGUUGACCUAUU 314 GTGTATGAT TGTATGAT GTATGAT hsa-miR-155 UUAAUGCUAAUCGUGAUAGGGGU 315 TTAGCATTA (SEQ ID NO:986) TAGCATTA (SEQ ID NO:4) AGCATTA (SEQ ID NO:987) hsa-miR-155* CUCCUACAUAUUAGCAUUAACA 316 TATGTAGGA ATGTAGGA TGTAGGA hsa-miR-15a UAGCAGCACAUAAUGGUUUGUG 317 TGTGCTGCT GTGCTGCT TGCTGCT hsa-miR-15a* CAGGCCAUAUUGUGCUGCCUCA 318 ATATGGCCT TATGGCCT ATGGCCT hsa-miR-15b UAGCAGCACAUCAUGGUUUACA 319 TGTGCTGCT GTGCTGCT TGCTGCT hsa-miR-15b* CGAAUCAUUAUUUGCUGCUCUA 320 TAATGATTC AATGATTC ATGATTC hsa-miR-16 UAGCAGCACGUAAAUAUUGGCG 321 CGTGCTGCT GTGCTGCT TGCTGCT hsa-miR-16-1* CCAGUAUUAACUGUGCUGCUGA 322 TTAATACTG TAATACTG AATACTG hsa-miR-16-2* CCAAUAUUACUGUGCUGCUUUA 323 GTAATATTG TAATATTG AATATTG hsa-miR-17 CAAAGUGCUUACAGUGCAGGUAG 324 AAGCACTTT AGCACTTT GCACTTT hsa-miR-17* ACUGCAGUGAAGGCACUUGUAG 325 TCACTGCAG CACTGCAG ACTGCAG hsa-miR-181a AACAUUCAACGCUGUCGGUGAGU 326 GTTGAATGT TTGAATGT TGAATGT hsa-miR-181a* ACCAUCGACCGUUGAUUGUACC 327 GGTCGATGG GTCGATGG TCGATGG hsa-miR-181a-2* ACCACUGACCGUUGACUGUACC 328 GGTCAGTGG GTCAGTGG TCAGTGG hsa-miR-181b AACAUUCAUUGCUGUCGGUGGGU 329 AATGAATGT ATGAATGT TGAATGT hsa-miR-181c AACAUUCAACCUGUCGGUGAGU 330 GTTGAATGT TTGAATGT TGAATGT hsa-miR-181c* AACCAUCGACCGUUGAGUGGAC 331 GTCGATGGT TCGATGGT CGATGGT hsa-miR-181d AACAUUCAUUGUUGUCGGUGGGU 332 AATGAATGT ATGAATGT TGAATGT hsa-miR-182 UUUGGCAAUGGUAGAACUCACACU 333 CATTGCCAA ATTGCCAA TTGCCAA hsa-miR-182* UGGUUCUAGACUUGCCAACUA 334 TCTAGAACC CTAGAACC TAGAACC hsa-miR-1825 UCCAGUGCCCUCCUCUCC 335 GGGCACTGG GGCACTGG GCACTGG hsa-miR-1826 AUUGAUCAUCGACACUUCGAACGCAAU 336 GATGATCAA ATGATCAA TGATCAA hsa-miR-1827 UGAGGCAGUAGAUUGAAU 337 TACTGCCTC ACTGCCTC CTGCCTC hsa-miR-183 UAUGGCACUGGUAGAAUUCACU 338 CAGTGCCAT AGTGCCAT GTGCCAT hsa-miR-183* GUGAAUUACCGAAGGGCCAUAA 339 GGTAATTCA GTAATTCA TAATTCA hsa-miR-184 UGGACGGAGAACUGAUAAGGGU 340 TCTCCGTCC CTCCGTCC TCCGTCC hsa-miR-185 UGGAGAGAAAGGCAGUUCCUGA 341 TTTCTCTCC TTCTCTCC TCTCTCC hsa-miR-185* AGGGGCUGGCUUUCCUCUGGUC 342 GCCAGCCCC CCAGCCCC CAGCCCC hsa-miR-186 CAAAGAAUUCUCCUUUUGGGCU 343 GAATTCTTT AATTCTTT ATTCTTT hsa-miR-186* GCCCAAAGGUGAAUUUUUUGGG 344 ACCTTTGGG CCTTTGGG CTTTGGG hsa-miR-187 UCGUGUCUUGUGUUGCAGCCGG 345 CAAGACACG AAGACACG AGACACG hsa-miR-187* GGCUACAACACAGGACCCGGGC 346 TGTTGTAGC GTTGTAGC TTGTAGC hsa-miR-188-3p CUCCCACAUGCAGGGUUUGCA 347 CATGTGGGA ATGTGGGA TGTGGGA hsa-miR-188-5p CAUCCCUUGCAUGGUGGAGGG 348 GCAAGGGAT CAAGGGAT AAGGGAT hsa-miR-18a UAAGGUGCAUCUAGUGCAGAUAG 349 ATGCACCTT TGCACCTT GCACCTT hsa-miR-18a* ACUGCCCUAAGUGCUCCUUCUGG 350 TTAGGGCAG TAGGGCAG AGGGCAG hsa-miR-18b UAAGGUGCAUCUAGUGCAGUUAG 351 ATGCACCTT TGCACCTT GCACCTT hsa-miR-18b* UGCCCUAAAUGCCCCUUCUGGC 352 ATTTAGGGC TTTAGGGC TTAGGGC hsa-miR-190 UGAUAUGUUUGAUAUAUUAGGU 353 AAACATATC AACATATC ACATATC hsa-miR-1908 CGGCGGGGACGGCGAUUGGUC 354 GTCCCCGCC TCCCCGCC CCCCGCC hsa-miR-1909 CGCAGGGGCCGGGUGCUCACCG 355 GGCCCCTGC GCCCCTGC CCCCTGC hsa-miR-1909* UGAGUGCCGGUGCCUGCCCUG 356 CCGCCACTC CGGCACTC GGCACTC hsa-miR-190b UGAUAUGUUUGAUAUUGGGUU 357 AAACATATC AACATATC ACATATC hsa-miR-191 CAACGGAAUCCCAAAAGCAGCUG 358 GATTCCGTT ATTCCGTT TTCCGTT hsa-miR-191* GCUGCGCUUGGAUUUCGUCCCC 359 CAAGCGCAG AAGCGCAG AGCGCAG hsa-miR-1910 CCAGUCCUGUGCCUGCCGCCU 360 ACAGGACTG CAGGACTG AGGACTG hsa-miR-1911 UGAGUACCGCCAUGUCUGUUGGG 361 GCGGTACTC CGGTACTC GGTACTC hsa-miR-1911* CACCAGGCAUUGUGGUCUCC 362 ATGCCTGGT TGCCTGGT GCCTGGT hsa-miR-1912 UACCCAGAGCAUGCAGUGUGAA 363 GCTCTGGGT CTCTGGGT TCTGGGT hsa-miR-1913 UCUGCCCCCUCCGCUGCUGCCA 364 AGGGGGCAG GGGGGCAG GGGGCAG hsa-miR-1914 CCCUGUGCCCGGCCCACUUCUG 365 GGGCACAGG GGCACAGG GCACAGG hsa-miR-1914* GGAGGGGUCCCGCACUGGGAGG 366 GGACCCCTC GACCCCTC ACCCCTC hsa-miR-1915 CCCCAGGGCGACGCGGCGGG 367 CGCCCTGGG GCCCTGGG CCCTGGG hsa-miR-1915* ACCUUGCCUUGCUGCCCGGGCC 368 AAGGCAAGG AGGCAAGG GGCAAGG hsa-miR-192 CUGACCUAUGAAUUGACAGCC 369 CATAGGTCA ATAGGTCA TAGGTCA hsa-miR-192* CUGCCAAUUCCAUAGGUCACAG 370 GAATTGGCA AATTGGCA ATTGGCA hsa-miR-193a-3p AACUGGCCUACAAAGUCCCAGU 371 TAGGCCAGT AGGCCAGT GGCCAGT hsa-miR-193a-5p UGGGUCUUUGCGGGCGAGAUGA 372 CAAAGACCC AAAGACCC AAGACCC hsa-miR-193b AACUGGCCCUCAAAGUCCCGCU 373 AGGGCCAGT GGGCCAGT GGCCAGT hsa-miR-193b* CGGGGUUUUGAGGGCGAGAUGA 374 CAAAACCCC AAAACCCC AAACCCC hsa-miR-194 UGUAACAGCAACUCCAUGUGGA 375 TGCTGTTAC GCTGTTAC CTGTTAC hsa-miR-194* CCAGUGGGGCUGCUGUUAUCUG 376 GCCCCACTG CCCCACTG CCCACTG hsa-miR-195 UAGCAGCACAGAAAUAUUGGC 377 TGTGCTGCT GTGCTGCT TGCTGCT hsa-miR-195* CCAAUAUUGGCUGUGCUGCUCC 378 CCAATATTG CAATATTG AATATTG hsa-miR-196a UAGGUAGUUUCAUGUUGUUGGG 379 AAACTACCT AACTACCT ACTACCT hsa-miR-196a* CGGCAACAAGAAACUGCCUGAG 380 CTTGTTGCC TTGTTGCC TGTTGCC hsa-miR-196b UAGGUAGUUUCCUGUUGUUGGG 381 AAACTACCT AACTACCT ACTACCT hsa-miR-197 UUCACCACCUUCUCCACCCAGC 382 AGGTGGTGA GGTGGTGA GTGGTGA hsa-miR-198 GGUCCAGAGGGGAGAUAGGUUC 383 CCTCTGGAC CTCTGGAC TCTGGAC hsa-miR-199a-5p CCCAGUGUUCAGACUACCUGUUC 384 GAACACTGG AACACTGG ACACTGG hsa-miR-199b-3p ACAGUAGUCUGCACAUUGGUUA 385 AGACTACTG GACTACTG ACTACTG hsa-miR-199b-5p CCCAGUGUUUAGACUAUCUGUUC 386 AAACACTGG AACACTGG ACACTGG hsa-miR-19a UGUGCAAAUCUAUGCAAAACUGA 387 GATTTGCAC ATTTGCAC TTTGCAC hsa-miR-19a* AGUUUUGCAUAGUUGCACUACA 388 ATGCAAAAC TGCAAAAC GCAAAAC hsa-miR-19b UGUGCAAAUCCAUGCAAAACUGA 389 GATTTGCAC (SEQ ID NO:992) ATTTGCAC (SEQ ID NO:20) TTTGCAC (SEQ ID NO:993) hsa-miR-19b-1* AGUUUUGCAGGUUUGCAUCCAGC 390 CTGCAAAAC TGCAAAAC GCAAAAC hsa-miR-19b-2* AGUUUUGCAGGUUUGCAUUUCA 391 CTGCAAAAC TGCAAAAC GCAAAAC hsa-miR-200a UAACACUGUCUGGUAACGAUGU 392 GACAGTGTT ACAGTGTT CAGTGTT hsa-miR-200a* CAUCUUACCGGACAGUGCUGGA 393 CGGTAAGAT GGTAAGAT GTAAGAT hsa-miR-200b UAAUACUGCCUGGUAAUGAUGA 394 GGCAGTATT GCAGTATT CAGTATT hsa-miR-200b* CAUCUUACUGGGCAGCAUUGGA 395 CAGTAAGAT AGTAAGAT GTAAGAT hsa-miR-200c UAAUACUGCCGGGUAAUGAUGGA 396 GGCAGTATT GCAGTATT CAGTATT hsa-miR-200c* CGUCUUACCCAGCAGUGUUUGG 397 GGGTAAGAC GGTAAGAC GTAAGAC hsa-miR-202 AGAGGUAUAGGGCAUGGGAA 398 CTATACCTC TATACCTC ATACCTC hsa-miR-202* UUCCUAUGCAUAUACUUCUUUG 399 TGCATAGGA GCATAGGA CATAGGA hsa-miR-203 GUGAAAUGUUUAGGACCACUAG 400 AACATTTCA ACATTTCA CATTTCA hsa-miR-204 UUCCCUUUGUCAUCCUAUGCCU 401 ACAAAGGGA CAAAGGGA AAAGGGA hsa-miR-205 UCCUUCAUUCCACCGGAGUCUG 402 GAATGAAGG AATGAAGG ATGAAGG hsa-miR-206 UGGAAUGUAAGGAAGUGUGUGG 403 TTACATTCC TACATTCC ACATTCC hsa-miR-208a AUAAGACGAGCAAAAAGCUUGU 404 CTCGTCTTA TCGTCTTA CGTCTTA hsa-miR-208b AUAAGACGAACAAAAGGUUUGU 405 TTCGTCTTA TCGTCTTA CGTCTTA hsa-miR-20a UAAAGUGCUUAUAGUGCAGGUAG 406 AAGCACTTT AGCACTTT GCACTTT hsa-miR-20a* ACUGCAUUAUGAGCACUUAAAG 407 ATAATGCAG TAATGCAG AATGCAG hsa-miR-20b CAAAGUGCUCAUAGUGCAGGUAG 408 GAGCACTTT AGCACTTT GCACTTT hsa-miR-20b* ACUGUAGUAUGGGCACUUCCAG 409 ATACTACAG TACTACAG ACTACAG hsa-miR-21 UAGCUUAUCAGACUGAUGUUGA 410 TGATAAGCT (SEQ ID NO:9) GATAAGCT (SEQ ID NO:2) ATAAGCT (SEQ ID NO:8) hsa-miR-21* CAACACCAGUCGAUGGGCUGU 411 ACTGGTGTT CTGGTGTT TGGTGTT hsa-miR-210 CUGUGCGUGUGACAGCGGCUGA 412 ACACGCACA CACGCACA ACGCACA hsa-miR-211 UUCCCUUUGUCAUCCUUCGCCU 413 ACAAAGGGA CAAAGGGA AAAGGGA hsa-miR-212 UAACAGUCUCCAGUCACGGCC 414 GAGACTGTT AGACTGTT GACTGTT hsa-miR-214 ACAGCAGGCACAGACAGGCAGU 415 TGCCTGCTG GCCTGCTG CCTGCTG hsa-miR-214* UGCCUGUCUACACUUGCUGUGC 416 TAGACAGGC AGACAGGC GACAGGC hsa-miR-215 AUGACCUAUGAAUUGACAGAC 417 CATAGGTCA ATAGGTCA TAGGTCA hsa-miR-216a UAAUCUCAGCUGGCAACUGUGA 418 GCTGAGATT CTGAGATT TGAGATT hsa-miR-216b AAAUCUCUGCAGGCAAAUGUGA 419 GCAGAGATT CAGAGATT AGAGATT hsa-miR-217 UACUGCAUCAGGAACUGAUUGGA 420 TGATGCAGT GATGCAGT ATGCAGT hsa-miR-218 UUGUGCUUGAUCUAACCAUGU 421 TCAAGCACA CAAGCACA 5AAGCACA hsa-miR-218-1* AUGGUUCCGUCAAGCACCAUGG 422 ACGGAACCA CGGAACCA GGAACCA hsa-miR-218-2* CAUGGUUCUGUCAAGCACCGCG 423 CAGAACCAT AGAACCAT GAACCAT hsa-miR-219-1-3p AGAGUUGAGUCUGGACGUCCCG 424 ACTCAACTC CTCAACTC TCAACTC hsa-miR-219-2-3p AGAAUUGUGGCUGGACAUCUGU 425 CCACAATTC CACAATTC ACAATTC hsa-miR-219-5p UGAUUGUCCAAACGCAAUUCU 426 TGGACAATC GGACAATC GACAATC hsa-miR-22 AAGCUGCCAGUUGAAGAACUGU 427 CTGGCAGCT TGGCAGCT GGCAGCT hsa-miR-22* AGUUCUUCAGUGGCAAGCUUUA 428 CTGAAGAAC TGAAGAAC GAAGAAC hsa-miR-220a CCACACCGUAUCUGACACUUU 429 TACGGTGTG ACGGTGTG CGGTGTG hsa-miR-220b CCACCACCGUGUCUGACACUU 430 ACGGTGGTG CGGTGGTG GGTGGTG hsa-miR-220c ACACAGGGCUGUUGUGAAGACU 431 AGCCCTGTG GCCCTGTG CCCTGTG hsa-miR-221 AGCUACAUUGUCUGCUGGGUUUC  432 CAATGTAGC (SEQ ID NO:995) AATGTAGC (SEQ ID NO:996) ATGTAGC (SEQ ID NO:23) hsa-miR-221* ACCUGGCAUACAAUGUAGAUUU 433 TATGCCAGG ATGCCAGG TGCCAGG hsa-miR-222 AGCUACAUCUGGCUACUGGGU 434 AGATGTAGC (SEQ ID NO:998) GATGTAGC (SEQ ID NO:999) ATGTAGC (SEQ ID NO:23) hsa-miR-222* CUCAGUAGCCAGUGUAGAUCCU 435 GGCTACTGA GCTACTGA CTACTGA hsa-miR-223 UGUCAGUUUGUCAAAUACCCCA 436 CAAACTGAC AAACTGAC AACTGAC hsa-miR-223* CGUGUAUUUGACAAGCUGAGUU 437 CAAATACAC AAATACAC AATACAC hsa-miR-224 CAAGUCACUAGUGGUUCCGUU 438 TAGTGACTT AGTGACTT GTGACTT hsa-miR-23a AUCACAUUGCCAGGGAUUUCC 439 GCAATGTGA CAATGTGA AATGTGA hsa-miR-23a* GGGGUUCCUGGGGAUGGGAUUU 440 CAGGAACCC AGGAACCC GGAACCC hsa-miR-23b AUCACAUUGCCAGGGAUUACC 441 GCAATGTGA CAATGTGA AATGTGA hsa-miR-23b* UGGGUUCCUGGCAUGCUGAUUU 442 CAGGAACCC AGGAACCC GGAACCC hsa-miR-24 UGGCUCAGUUCAGCAGGAACAG 443 AACTGAGCC ACTGAGCC CTGAGCC hsa-miR-24-1* UGCCUACUGAGCUGAUAUCAGU 444 TCAGTAGGC CAGTAGGC AGTAGGC hsa-miR-24-2* UGCCUACUGAGCUGAAACACAG 445 TCAGTAGGC CAGTAGGC AGTAGGC hsa-miR-25 CAUUGCACUUGUCUCGGUCUGA 446 AAGTGCAAT AGTGCAAT GTGCAAT hsa-miR-25* AGGCGGAGACUUGGGCAAUUG 447 GTCTCCGCC TCTCCGCC CTCCGCC hsa-miR-26a UUCAAGUAAUCCAGGAUAGGCU 448 ATTACTTGA TTACTTGA TACTTGA hsa-miR-26a-1* CCUAUUCUUGGUUACUUGCACG 449 CAAGAATAG AAGAATAG AGAATAG hsa-miR-26a-2* CCUAUUCUUGAUUACUUGUUUC 450 CAAGAATAG AAGAATAG AGAATAG hsa-miR-26b UUCAAGUAAUUCAGGAUAGGU 451 ATTACTTGA TTACTTGA TACTTGA hsa-miR-26b* CCUGUUCUCCAUUACUUGGCUC 452 GGAGAACAG GAGAACAG AGAACAG hsa-miR-27a UUCACAGUGGCUAAGUUCCGC 453 CCACTGTGA CACTGTGA ACTGTGA hsa-miR-27a* AGGGCUUAGCUGCUUGUGAGCA 454 GCTAAGCCC CTAAGCCC TAAGCCC hsa-miR-27b UUCACAGUGGCUAAGUUCUGC 455 CCACTGTGA CACTGTGA ACTGTGA hsa-miR-27b* AGAGCUUAGCUGAUUGGUGAAC 456 GCTAAGCTC CTAAGCTC TAAGCTC hsa-miR-28-3p CACUAGAUUGUGAGCUCCUGGA 457 CAATCTAGT AATCTAGT ATCTAGT hsa-miR-28-5p AAGGAGCUCACAGUCUAUUGAG 458 TGAGCTCCT GAGCTCCT AGCTCCT hsa-miR-296-3p GAGGGUUGGGUGGAGGCUCUCC 459 CCCAACCCT CCAACCCT CAACCCT hsa-miR-296-5p AGGGCCCCCCCUCAAUCCUGU 460 GGGGGGCCC GGGGGCCC GGGGCCC hsa-miR-297 AUGUAUGUGUGCAUGUGCAUG 461 ACACATACA CACATACA ACATACA hsa-miR-298 AGCAGAAGCAGGGAGGUUCUCCCA 462 TGCTTCTGC GCTTCTGC CTTCTGC hsa-miR-299-3p UAUGUGGGAUGGUAAACCGCUU 463 ATCCCACAT TCCCACAT CCCACAT hsa-miR-299-5p UGGUUUACCGUCCCACAUACAU 464 CGGTAAACC GGTAAACC GTAAACC hsa-miR-29a UAGCACCAUCUGAAAUCGGUUA 465 GATGGTGCT ATGGTGCT TGGTGCT hsa-miR-29a* ACUGAUUUCUUUUGGUGUUCAG 466 AGAAATCAG GAAATCAG AAATCAG hsa-miR-29b UAGCACCAUUUGAAAUCAGUGUU 467 AATGGTGCT ATGGTGCT TGGTGCT hsa-miR-29b-1* GCUGGUUUCAUAUGGUGGUUUAGA 468 TGAAACCAG GAAACCAG AAACCAG hsa-miR-29b-2* CUGGUUUCACAUGGUGGCUUAG 469 GTGAAACCA TGAAACCA GAAACCA hsa-miR-29c UAGCACCAUUUGAAAUCGGUUA 470 AATGGTGCT ATGGTGCT TGGTGCT hsa-miR-29c* UGACCGAUUUCUCCUGGUGUUC 471 AAATCGGTC AATCGGTC ATCGGTC hsa-miR-300 UAUACAAGGGCAGACUCUCUCU 472 CCCTTGTAT CCTTGTAT CTTGTAT hsa-miR-301a CAGUGCAAUAGUAUUGUCAAAGC  473 TATTGCACT ATTGCACT TTGCACT hsa-miR-301b CAGUGCAAUGAUAUUGUCAAAGC 474 CATTGCACT ATTGCACT TTGCACT hsa-miR-302a UAAGUGCUUCCAUGUUUUGGUGA 475 GAAGCACTT AAGCACTT AGCACTT hsa-miR-302a* ACUUAAACGUGGAUGUACUUGCU 476 ACGTTTAAG CGTTTAAG GTTTAAG hsa-miR-302b UAAGUGCUUCCAUGUUUUAGUAG 477 GAAGCACTT AAGCACTT AGCACTT hsa-miR-302b* ACUUUAACAUGGAAGUGCUUUC 478 ATGTTAAAG TGTTAAAG GTTAAAG hsa-miR-302c UAAGUGCUUCCAUGUUUCAGUGG 479 GAAGCACTT AAGCACTT AGCACTT hsa-miR-302c* UUUAACAUGGGGGUACCUGCUG 480 CCATGTTAA CATGTTAA ATGTTAA hsa-miR-302d UAAGUGCUUCCAUGUUUGAGUGU 481 GAAGCACTT AAGCACTT AGCACTT hsa-miR-302d* ACUUUAACAUGGAGGCACUUGC 482 ATGTTAAAG TGTTAAAG GTTAAAG hsa-miR-302e UAAGUGCUUCCAUGCUU 483 GAAGCACTT AAGCACTT AGCACTT hsa-miR-3028 UAAUUGCUUCCAUGUUU 484 GAAGCAATT AAGCAATT AGCAATT hsa-miR-30a UGUAAACAUCCUCGACUGGAAG 485 GATGTTTAC ATGTTTAC TGTTTAC hsa-miR-30a* CUUUCAGUCGGAUGUUUGCAGC 486 CGACTGAAA GACTGAAA ACTGAAA hsa-miR-30b UGUAAACAUCCUACACUCAGCU 487 GATGTTTAC ATGTTTAC TGTTTAC hsa-miR-30b* CUGGGAGGUGGAUGUUUACUUC 488 CACCTCCCA ACCTCCCA CCTCCCA hsa-miR-30c UGUAAACAUCCUACACUCUCAGC 489 GATGTTTAC ATGTTTAC TGTTTAC hsa-miR-30c-1*  CUGGGAGAGGGUUGUUUACUCC 490 CCTCTCCCA CTCTCCCA TCTCCCA hsa-miR-30c-2*  CUGGGAGAAGGCUGUUUACUCU 491 CTTCTCCCA TTCTCCCA TCTCCCA hsa-miR-30d UGUAAACAUCCCCGACUGGAAG 492 GATGTTTAC ATGTTTAC TGTTTAC hsa-miR-30d* CUUUCAGUCAGAUGUUUGCUGC 493 TGACTGAAA GACTGAAA ACTGAAA hsa-miR-30e UGUAAACAUCCUUGACUGGAAG 494 GATGTTTAC ATGTTTAC TGTTTAC hsa-miR-30e* CUUUCAGUCGGAUGUUUACAGC 495 CGACTGAAA GACTGAAA ACTGAAA hsa-miR-31 AGGCAAGAUGCUGGCAUAGCU 496 CATCTTGCC ATCTTGCC TCTTGCC hsa-miR-31* UGCUAUGCCAACAUAUUGCCAU 497 TGGCATAGC GGCATAGC GCATAGC hsa-miR-32 UAUUGCACAUUACUAAGUUGCA 498 ATGTGCAAT TGTGCAAT GTGCAAT hsa-miR-32* CAAUUUAGUGUGUGUGAUAUUU 499 CACTAAATT ACTAAATT CTAAATT hsa-miR-320a AAAAGCUGGGUUGAGAGGGCGA 500 CCCAGCTTT CCAGCTTT CAGCTTT hsa-miR-320b AAAAGCUGGGUUGAGAGGGCAA 501 CCCAGCTTT CCAGCTTT CAGCTTT hsa-miR-320c AAAAGCUGGGUUGAGAGGGU 502 CCCAGCTTT CCAGCTTT CAGCTTT hsa-miR-320d AAAAGCUGGGUUGAGAGGA 503 CCCAGCTTT CCAGCTTT CAGCTTT hsa-miR-323-3p CACAUUACACGGUCGACCUCU 504 GTGTAATGT TGTAATGT GTAATGT hsa-miR-323-5p AGGUGGUCCGUGGCGCGUUCGC 505 CGGACCACC GGACCACC GACCACC hsa-miR-324-3p ACUGCCCCAGGUGCUGCUGG 506 CTGGGGCAG TGGGGCAG GGGGCAG hsa-miR-324-5p CGCAUCCCCUAGGGCAUUGGUGU 507 AGGGGATGC GGGGATGC GGGATGC hsa-miR-325 CCUAGUAGGUGUCCAGUAAGUGU 508 ACCTACTAG CCTACTAG CTACTAG hsa-miR-326 CCUCUGGGCCCUUCCUCCAG 509 GGCCCAGAG GCCCAGAG CCCAGAG hsa-miR-328 CUGGCCCUCUCUGCCCUUCCGU 510 AGAGGGCCA GAGGGCCA AGGGCCA hsa-miR-329 AACACACCUGGUUAACCUCUUU 511 CAGGTGTGT AGGTGTGT GGTGTGT hsa-miR-330-3p GCAAAGCACACGGCCUGCAGAGA 512 TGTGCTTTG GTGCTTTG TGCTTTG hsa-miR-330-5p UCUCUGGGCCUGUGUCUUAGGC 513 GGCCCAGAG GCCCAGAG CCCAGAG hsa-miR-331-3p GCCCCUGGGCCUAUCCUAGAA 514 GCCCAGGGG CCCAGGGG CCAGGGG hsa-miR-331-5p CUAGGUAUGGUCCCAGGGAUCC 515 CCATACCTA CATACCTA ATACCTA hsa-miR-335 UCAAGAGCAAUAACGAAAAAUGU 516 TTGCTCTTG TGCTCTTG GCTCTTG hsa-miR-335* UUUUUCAUUAUUGCUCCUGACC 517 TAATGAAAA AATGAAAA ATGAAAA hsa-miR-337-3p CUCCUAUAUGAUGCCUUUCUUC 518 CATATAGGA ATATAGGA TATAGGA hsa-miR-337-5p GAACGGCUUCAUACAGGAGUU 519 GAAGCCGTT AAGCCGTT AGCCGTT hsa-miR-338-3p UCCAGCAUCAGUGAUUUUGUUG 520 TGATGCTGG GATGCTGG ATGCTGG hsa-miR-338-5p AACAAUAUCCUGGUGCUGAGUG 521 GGATATTGT GATATTGT ATATTGT hsa-miR-339-3p UGAGCGCCUCGACGACAGAGCCG 522 GAGGCGCTC AGGCGCTC GGCGCTC hsa-miR-339-5p UCCCUGUCCUCCAGGAGCUCACG 523 AGGACAGGG GGACAGGG GACAGGG hsa-miR-33a GUGCAUUGUAGUUGCAUUGCA 524 TACAATGCA ACAATGCA CAATGCA hsa-miR-33a* CAAUGUUUCCACAGUGCAUCAC 525 GGAAACATT GAAACATT AAACATT hsa-miR-33b GUGCAUUGCUGUUGCAUUGC 526 AGCAATGCA GCAATGCA CAATGCA hsa-miR-33b* CAGUGCCUCGGCAGUGCAGCCC 527 CGAGGCACT GAGGCACT AGGCACT hsa-miR-340 UUAUAAAGCAAUGAGACUGAUU 528 TGCTTTATA GCTTTATA CTTTATA hsa-miR-340* UCCGUCUCAGUUACUUUAUAGC 529 CTGAGACGG TGAGACGG GAGACGG hsa-miR-342-3p UCUCACACAGAAAUCGCACCCGU 530 CTGTGTGAG TGTGTGAG GTGTGAG hsa-miR-342-5p AGGGGUGCUAUCUGUGAUUGA 531 TAGCACCCC AGCACCCC GCACCCC hsa-miR-345 GCUGACUCCUAGUCCAGGGCUC 532 AGGAGTCAG GGAGTCAG GAGTCAG hsa-miR-346 UGUCUGCCCGCAUGCCUGCCUCU 533 CGGGCAGAC GGGCAGAC GGCAGAC hsa-miR-34a UGGCAGUGUCUUAGCUGGUUGU 534 GACACTGCC ACACTGCC CACTGCC hsa-miR-34a* CAAUCAGCAAGUAUACUGCCCU 535 TTGCTGATT TGCTGATT GCTGATT hsa-miR-34b CAAUCACUAACUCCACUGCCAU 536 TTAGTGATT TAGTGATT AGTGATT hsa-miR-34b* UAGGCAGUGUCAUUAGCUGAUUG 537 ACACTGCCT CACTGCCT ACTGCCT hsa-miR-34c-3p AAUCACUAACCACACGGCCAGG 538 GTTAGTGAT TTAGTGAT TAGTGAT hsa-miR-34c-5p AGGCAGUGUAGUUAGCUGAUUGC 539 TACACTGCC ACACTGCC CACTGCC hsa-miR-361-3p UCCCCCAGGUGUGAUUCUGAUUU 540 ACCTGGGGG CCTGGGGG CTGGGGG hsa-miR-361-5p UUAUCAGAAUCUCCAGGGGUAC 541 ATTCTGATA TTCTGATA TCTGATA hsa-miR-362-3p AACACACCUAUUCAAGGAUUCA 542 TAGGTGTGT AGGTGTGT GGTGTGT hsa-miR-362-5p AAUCCUUGGAACCUAGGUGUGAGU 543 TCCAAGGAT CCAAGGAT CAAGGAT hsa-miR-363 AAUUGCACGGUAUCCAUCUGUA 544 CCGTGCAAT CGTGCAAT GTGCAAT hsa-miR-363* CGGGUGGAUCACGAUGCAAUUU 545 GATCCACCC  ATCCACCC TCCACCC hsa-miR-365 UAAUGCCCCUAAAAAUCCUUAU 546 AGGGGCATT GGGGCATT GGGCATT hsa-miR-367 AAUUGCACUUUAGCAAUGGUGA 547 AAGTGCAAT AGTGCAAT GTGCAAT hsa-miR-367* ACUGUUGCUAAUAUGCAACUCU 548 TAGCAACAG AGCAACAG GCAACAG hsa-miR-369-3p AAUAAUACAUGGUUGAUCUUU 549 ATGTATTAT TGTATTAT GTATTAT hsa-miR-369-5p AGAUCGACCGUGUUAUAUUCGC 550 CGGTCGATC GGTCGATC GTCGATC hsa-miR-370 GCCUGCUGGGGUGGAACCUGGU 551 CCCAGCAGG CCAGCAGG CAGCAGG hsa-miR-371-3p AAGUGCCGCCAUCUUUUGAGUGU 552 GGCGGCACT GCGGCACT CGGCACT hsa-miR-371-5p ACUCAAACUGUGGGGGCACU 553 CAGTTTGAG AGTTTGAG GTTTGAG hsa-miR-372 AAAGUGCUGCGACAUUUGAGCGU 554 GCAGCACTT CAGCACTT AGCACTT hsa-miR-373 GAAGUGCUUCGAUUUUGGGGUGU 555 GAAGCACTT AAGCACTT AGCACTT hsa-miR-373* ACUCAAAAUGGGGGCGCUUUCC 556 CATTTTGAG ATTTTGAG TTTTGAG hsa-miR-374a UUAUAAUACAACCUGAUAAGUG 557 TGTATTATA GTATTATA TATTATA hsa-miR-374a* CUUAUCAGAUUGUAUUGUAAUU 558 ATCTGATAA TCTGATAA CTGATAA hsa-miR-374b AUAUAAUACAACCUGCUAAGUG 559 TGTATTATA GTATTATA TATTATA hsa-miR-374b* CUUAGCAGGUUGUAUUAUCAUU 560 ACCTGCTAA CCTGCTAA CTGCTAA hsa-miR-375 UUUGUUCGUUCGGCUCGCGUGA 561 AACGAACAA ACGAACAA CGAACAA hsa-miR-376a AUCAUAGAGGAAAAUCCACGU 562 CCTCTATGA CTCTATGA TCTATGA hsa-miR-376a* GUAGAUUCUCCUUCUAUGAGUA 563 GAGAATCTA AGAATCTA GAATCTA hsa-miR-376b AUCAUAGAGGAAAAUCCAUGUU 564 CCTCTATGA CTCTATGA TCTATGA hsa-miR-376c AACAUAGAGGAAAUUCCACGU 565 CCTCTATGT CTCTATGT TCTATGT hsa-miR-377 AUCACACAAAGGCAACUUUUGU 566 TTTGTGTGA TTGTGTGA TGTGTGA hsa-miR-377* AGAGGUUGCCCUUGGUGAAUUC 567 GGCAACCTC GCAACCTC CAACCTC hsa-miR-378 ACUGGACUUGGAGUCAGAAGG 568 CAAGTCCAG AAGTCCAG AGTCCAG hsa-miR-378* CUCCUGACUCCAGGUCCUGUGU 569 GAGTCAGGA AGTCAGGA GTCAGGA hsa-miR-379 UGGUAGACUAUGGAACGUAGG 570 TAGTCTACC AGTCTACC GTCTACC hsa-miR-379* UAUGUAACAUGGUCCACUAACU 571 ATGTTACAT TGTTACAT GTTACAT hsa-miR-380 UAUGUAAUAUGGUCCACAUCUU 572 ATATTACAT TATTACAT ATTACAT hsa-miR-380* UGGUUGACCAUAGAACAUGCGC 573 TGGTCAACC GGTCAACC GTCAACC hsa-miR-381 UAUACAAGGGCAAGCUCUCUGU 574 CCCTTGTAT CCTTGTAT CTTGTAT hsa-miR-382 GAAGUUGUUCGUGGUGGAUUCG 575 GAACAACTT AACAACTT ACAACTT hsa-miR-383 AGAUCAGAAGGUGAUUGUGGCU 576 CTTCTGATC TTCTGATC TCTGATC hsa-miR-384 AUUCCUAGAAAUUGUUCAUA 577 TTCTAGGAA TCTAGGAA CTAGGAA hsa-miR-409-3p GAAUGUUGCUCGGUGAACCCCU 578 AGCAACATT GCAACATT CAACATT hsa-miR-409-5p AGGUUACCCGAGCAACUUUGCAU 579 CGGGTAACC GGGTAACC GGTAACC hsa-miR-410 AAUAUAACACAGAUGGCCUGU 580 GTGTTATAT TGTTATAT GTTATAT hsa-miR-411 UAGUAGACCGUAUAGCGUACG 581 CGGTCTACT GGTCTACT GTCTACT hsa-miR-411* UAUGUAACACGGUCCACUAACC 582 GTGTTACAT TGTTACAT GTTACAT hsa-miR-412 ACUUCACCUGGUCCACUAGCCGU 583 CAGGTGAAG AGGTGAAG GGTGAAG hsa-miR-421 AUCAACAGACAUUAAUUGGGCGC  584 GTCTGTTGA TCTGTTGA CTGTTGA hsa-miR-422a ACUGGACUUAGGGUCAGAAGGC 585 TAAGTCCAG AAGTCCAG AGTCCAG hsa-miR-423-3p AGCUCGGUCUGAGGCCCCUCAGU  586 AGACCGAGC GACCGAGC ACCGAGC hsa-miR-423-5p UGAGGGGCAGAGAGCGAGACUUU  587 CTGCCCCTC TGCCCCTC GCCCCTC hsa-miR-424 CAGCAGCAAUUCAUGUUUUGAA 588 ATTGCTGCT TTGCTGCT TGCTGCT hsa-miR-424* CAAAACGUGAGGCGCUGCUAU 589 TCACGTTTT CACGTTTT ACGTTTT hsa-miR-425 AAUGACACGAUCACUCCCGUUGA  590 TCGTGTCAT CGTGTCAT GTGTCAT hsa-miR-425* AUCGGGAAUGUCGUGUCCGCCC 591 CATTCCCGA ATTCCCGA TTCCCGA hsa-miR-429 UAAUACUGUCUGGUAAAACCGU 592 GACAGTATT ACAGTATT CAGTATT hsa-miR-431 UGUCUUGCAGGCCGUCAUGCA 593 CTGCAAGAC TGCAAGAC GCAAGAC hsa-miR-431* CAGGUCGUCUUGCAGGGCUUCU 594 AGACGACCT GACGACCT ACGACCT hsa-miR-432 UCUUGGAGUAGGUCAUUGGGUGG  595 TACTCCAAG ACTCCAAG CTCCAAG hsa-miR-432* CUGGAUGGCUCCUCCAUGUCU 596 AGCCATCCA GCCATCCA CCATCCA hsa-miR-433 AUCAUGAUGGGCUCCUCGGUGU 597 CCATCATGA CATCATGA ATCATGA hsa-miR-448 UUGCAUAUGUAGGAUGUCCCAU 598 ACATATGCA CATATGCA ATATGCA hsa-miR-449a UGGCAGUGUAUUGUUAGCUGGU 599 TACACTGCC ACACTGCC CACTGCC hsa-miR-449b AGGCAGUGUAUUGUUAGCUGGC 600 TACACTGCC ACACTGCC CACTGCC hsa-miR-450a UUUUGCGAUGUGUUCCUAAUAU 601 CATCGCAAA ATCGCAAA TCGCAAA hsa-miR-450b-3p UUGGGAUCAUUUUGCAUCCAUA 602 ATGATCCCA TGATCCCA GATCCCA hsa-miR-450b-5p UUUUGCAAUAUGUUCCUGAAUA 603 TATTGCAAA ATTGCAAA TTGCAAA hsa-miR-451 AAACCGUUACCAUUACUGAGUU 604 GTAACGGTT TAACGGTT AACGGTT hsa-miR-452 AACUGUUUGCAGAGGAAACUGA 605 GCAAACAGT CAAACAGT AAACAGT hsa-miR-452* CUCAUCUGCAAAGAAGUAAGUG 606 TGCAGATGA GCAGATGA CAGATGA hsa-miR-453 AGGUUGUCCGUGGUGAGUUCGCA  607 CGGACAACC GGACAACC GACAACC hsa-miR-454 UAGUGCAAUAUUGCUUAUAGGGU  608 TATTGCACT ATTGCACT TTGCACT hsa-miR-454* ACCCUAUCAAUAUUGUCUCUGC 609 TTGATAGGG TGATAGGG GATAGGG hsa-miR-455-3p GCAGUCCAUGGGCAUAUACAC 610 CATGGACTG ATGGACTG TGGACTG hsa-miR-455-5p UAUGUGCCUUUGGACUACAUCG 611 AAGGCACAT AGGCACAT GGCACAT hsa-miR-483-3p UCACUCCUCUCCUCCCGUCUU 612 AGAGGAGTG GAGGAGTG AGGAGTG hsa-miR-483-5p AAGACGGGAGGAAAGAAGGGAG 613 CTCCCGTCT TCCCGTCT CCCGTCT hsa-miR-484 UCAGGCUCAGUCCCCUCCCGAU 614 CTGAGCCTG TGAGCCTG GAGCCTG hsa-miR-485-3p GUCAUACACGGCUCUCCUCUCU 615 CGTGTATGA GTGTATGA TGTATGA hsa-miR-485-5p AGAGGCUGGCCGUGAUGAAUUC 616 GCCAGCCTC CCAGCCTC CAGCCTC hsa-miR-486-3p CGGGGCAGCUCAGUACAGGAU 617 AGCTGCCCC GCTGCCCC CTGCCCC hsa-miR-486-5p UCCUGUACUGAGCUGCCCCGAG 618 CAGTACAGG AGTACAGG GTACAGG hsa-miR-487a AAUCAUACAGGGACAUCCAGUU 619 CTGTATGAT TGTATGAT GTATGAT hsa-miR-487b AAUCGUACAGGGUCAUCCACUU 620 CTGTACGAT TGTACGAT GTACGAT hsa-miR-488 UUGAAAGGCUAUUUCUUGGUC 621 AGCCTTTCA GCCTTTCA CCTTTCA hsa-miR-488* CCCAGAUAAUGGCACUCUCAA 622 ATTATCTGG TTATCTGG TATCTGG hsa-miR-489 GUGACAUCACAUAUACGGCAGC 623 GTGATGTCA TGATGTCA GATGTCA hsa-miR-490-3p CAACCUGGAGGACUCCAUGCUG 624 CTCCAGGTT TCCAGGTT CCAGGTT hsa-miR-490-5p CCAUGGAUCUCCAGGUGGGU 625 AGATCCATG GATCCATG ATCCATG hsa-miR-491-3p CUUAUGCAAGAUUCCCUUCUAC 626 CTTGCATAA TTGCATAA TGCATAA hsa-miR-491-5p AGUGGGGAACCCUUCCAUGAGG 627 GTTCCCCAC TTCCCCAC TCCCCAC hsa-miR-492 AGGACCUGCGGGACAAGAUUCUU  628 CGCAGGTCC GCAGGTCC CAGGTCC hsa-miR-493 UGAAGGUCUACUGUGUGCCAGG 629 TAGACCTTC AGACCTTC GACCTTC hsa-miR-493* UUGUACAUGGUAGGCUUUCAUU 630 CCATGTACA CATGTACA ATGTACA hsa-miR-494 UGAAACAUACACGGGAAACCUC 631 GTATGTTTC TATGTTTC ATGTTTC hsa-miR-495 AAACAAACAUGGUGCACUUCUU 632 ATGTTTGTT TGTTTGTT GTTTGTT hsa-miR-496 UGAGUAUUACAUGGCCAAUCUC 633 GTAATACTC TAATACTC AATACTC hsa-miR-497 CAGCAGCACACUGUGGUUUGU 634 TGTGCTGCT GTGCTGCT TGCTGCT hsa-miR-497* CAAACCACACUGUGGUGUUAGA 635 GTGTGGTTT TGTGGTTT GTGGTTT hsa-miR-498 UUUCAAGCCAGGGGGCGUUUUUC  636 TGGCTTGAA GGCTTGAA GCTTGAA hsa-miR-499-3p AACAUCACAGCAAGUCUGUGCU 637 CTGTGATGT TGTGATGT GTGATGT hsa-miR-499-5p UUAAGACUUGCAGUGAUGUUU 638 CAAGTCTTA AAGTCTTA AGTCTTA hsa-miR-500 UAAUCCUUGCUACCUGGGUGAGA 639 GCAAGGATT CAAGGATT AAGGATT hsa-miR-500* AUGCACCUGGGCAAGGAUUCUG 640 CCAGGTGCA CAGGTGCA AGGTGCA hsa-miR-501-3p AAUGCACCCGGGCAAGGAUUCU 641 CGGGTGCAT GGGTGCAT GGTGCAT hsa-miR-501-5p AAUCCUUUGUCCCUGGGUGAGA 642 ACAAAGGAT CAAAGGAT AAAGGAT hsa-miR-502-3p AAUGCACCUGGGCAAGGAUUCA 643 CAGGTGCAT AGGTGCAT GGTGCAT hsa-miR-502-5p AUCCUUGCUAUCUGGGUGCUA 644 TAGCAAGGA AGCAAGGA GCAAGGA hsa-miR-503 UAGCAGCGGGAACAGUUCUGCAG 645 CCCGCTGCT CCGCTGCT CGCTGCT hsa-miR-504 AGACCCUGGUCUGCACUCUAUC 646 ACCAGGGTC CCAGGGTC CAGGGTC hsa-miR-505 CGUCAACACUUGCUGGUUUCCU 647 AGTGTTGAC GTGTTGAC TGTTGAC hsa-miR-505* GGGAGCCAGGAAGUAUUGAUGU 648 CCTGGCTCC CTGGCTCC TGGCTCC hsa-miR-506 UAAGGCACCCUUCUGAGUAGA 649 GGGTGCCTT GGTGCCTT GTGCCTT hsa-miR-507 UUUUGCACCUUUUGGAGUGAA 650 AGGTGCAAA GGTGCAAA GTGCAAA hsa-miR-508-3p UGAUUGUAGCCUUUUGGAGUAGA 651 GCTACAATC CTACAATC TACAATC hsa-miR-508-5p UACUCCAGAGGGCGUCACUCAUG 652 CTCTGGAGT TCTGGAGT CTGGAGT hsa-miR-509-3-5p UACUGCAGACGUGGCAAUCAUG 653 GTCTGCAGT TCTGCAGT CTGCAGT hsa-miR-509-3p UGAUUGGUACGUCUGUGGGUAG 654 GTACCAATC TACCAATC ACCAATC hsa-miR-509-5p UACUGCAGACAGUGGCAAUCA 655 GTCTGCAGT TCTGCAGT CTGCAGT hsa-miR-510 UACUCAGGAGAGUGGCAAUCAC 656 CTCCTGAGT TCCTGAGT CCTGAGT hsa-miR-511 GUGUCUUUUGCUCUGCAGUCA 657 CAAAAGACA AAAAGACA AAAGACA hsa-miR-512-3p AAGUGCUGUCAUAGCUGAGGUC 658 GACAGCACT ACAGCACT CAGCACT hsa-miR-512-5p CACUCAGCCUUGAGGGCACUUUC 659 AGGCTGAGT GGCTGAGT GCTGAGT hsa-miR-513a-3p UAAAUUUCACCUUUCUGAGAAGG 660 GTGAAATTT TGAAATTT GAAATTT hsa-miR-513a-5p UUCACAGGGAGGUGUCAU 661 TCCCTGTGA CCCTGTGA CCTGTGA hsa-miR-513b UUCACAAGGAGGUGUCAUUUAU 662 TCCTTGTGA CCTTGTGA CTTGTGA hsa-miR-513c UUCUCAAGGAGGUGUCGUUUAU 663 TCCTTGAGA CCTTGAGA CTTGAGA hsa-miR-514 AUUGACACUUCUGUGAGUAGA 664 AAGTGTCAA AGTGTCAA GTGTCAA hsa-miR-515-3p GAGUGCCUUCUUUUGGAGCGUU 665 GAAGGCACT AGGCACT AGGCACT hsa-miR-515-5p UUCUCCAAAAGAAAGCACUUUCUG 666 TTTTGGAGA TTTGGAGA TTGGAGA hsa-miR-516a-3p UGCUUCCUUUCAGAGGGU 667 AAAGGAAGC AAGGAAGC AGGAAGC hsa-miR-516a-5p UUCUCGAGGAAAGAAGCACUUUC 668 TCCTCGAGA CCTCGAGA CTCGAGA hsa-miR-516b AUCUGGAGGUAAGAAGCACUUU 669 ACCTCCAGA CCTCCAGA CTCCAGA hsa-miR-517* CCUCUAGAUGGAAGCACUGUCU 670 CATCTAGAG ATCTAGAG TCTAGAG hsa-miR-517a AUCGUGCAUCCCUUUAGAGUGU 671 GATGCACGA ATGCACGA TGCACGA hsa-miR-517b UCGUGCAUCCCUUUAGAGUGUU 672 GGATGCACG GATGCACG ATGCACG hsa-miR-517c AUCGUGCAUCCUUUUAGAGUGU 673 GATGCACGA ATGCACGA TGCACGA hsa-miR-518a-3p GAAAGCGCUUCCCUUUGCUGGA 674 AAGCGCTTT AGCGCTTT GCGCTTT hsa-miR-518b CAAAGCGCUCCCCUUUAGAGGU 675 GAGCGCTTT AGCGCTTT GCGCTTT hsa-miR-518c CAAAGCGCUUCUCUUUAGAGUGU 676 AAGCGCTTT AGCGCTTT GCGCTTT hsa-miR-518c* UCUCUGGAGGGAAGCACUUUCUG 677 CCTCCAGAG CTCCAGAG TCCAGAG hsa-miR-518d-3p CAAAGCGCUUCCCUUUGGAGC 678 AAGCGCTTT AGCGCTTT GCGCTTT hsa-miR-518d-5p CUCUAGAGGGAAGCACUUUCUG 679 CCCTCTAGA CCTCTAGA CTCTAGA hsa-miR-518e AAAGCGCUUCCCUUCAGAGUG 680 GAAGCGCTT AAGCGCTT AGCGCTT hsa-miR-518f GAAAGCGCUUCUCUUUAGAGG 681 AAGCGCTTT AGCGCTTT GCGCTTT hsa-miR-518f* CUCUAGAGGGAAGCACUUUCUC 682 CCCTCTAGA CCTCTAGA CTCTAGA hsa-miR-519a AAAGUGCAUCCUUUUAGAGUGU 683 GATGCACTT ATGCACTT TGCACTT hsa-miR-519a* CUCUAGAGGGAAGCGCUUUCUG 684 CCCTCTAGA CCTCTAGA CTCTAGA hsa-miR-519b-3p AAAGUGCAUCCUUUUAGAGGUU 685 GATGCACTT ATGCACTT TGCACTT hsa-miR-519c-3p AAAGUGCAUCUUUUUAGAGGAU 686 GATGCACTT ATGCACTT TGCACTT hsa-miR-519d CAAAGUGCCUCCCUUUAGAGUG 687 AGGCACTTT GGCACTTT GCACTTT hsa-miR-519e AAGUGCCUCCUUUUAGAGUGUU 688 GGAGGCACT GAGGCACT AGGCACT hsa-miR-519e* UUCUCCAAAAGGGAGCACUUUC 689 TTTTGGAGA TTTGGAGA TTGGAGA hsa-miR-520a-3p AAAGUGCUUCCCUUUGGACUGU 690 GAAGCACTT AAGCACTT AGCACTT hsa-miR-520a-5p CUCCAGAGGGAAGUACUUUCU 691 CCCTCTGGA CCTCTGGA CTCTGGA hsa-miR-520b AAAGUGCUUCCUUUUAGAGGG 692 GAAGCACTT AAGCACTT AGCACTT hsa-miR-520c-3p AAAGUGCUUCCUUUUAGAGGGU 693 GAAGCACTT AAGCACTT AGCACTT hsa-miR-520d-3p AAAGUGCUUCUCUUUGGUGGGU 694 GAAGCACTT AAGCACTT AGCACTT hsa-miR-520d-5p CUACAAAGGGAAGCCCUUUC 695 CCCTTTGTA CCTTTGTA CTTTGTA hsa-miR-520e AAAGUGCUUCCUUUUUGAGGG 696 GAAGCACTT AAGCACTT AGCACTT hsa-miR-520f AAGUGCUUCCUUUUAGAGGGUU 697 GGAAGCACT GAAGCACT AAGCACT hsa-miR-520g ACAAAGUGCUUCCCUUUAGAGUGU 698 AGCACTTTG GCACTTTG CACTTTG hsa-miR-520h ACAAAGUGCUUCCCUUUAGAGU 699 AGCACTTTG GCACTTTG CACTTTG hsa-miR-521 AACGCACUUCCCUUUAGAGUGU 700 GAAGTGCGT AAGTGCGT AGTGCGT hsa-miR-522 AAAAUGGUUCCCUUUAGAGUGU 701 GAACCATTT AACCATTT ACCATTT hsa-miR-523 GAACGCGCUUCCCUAUAGAGGGU 702 AAGCGCGTT AGCGCGTT GCGCGTT hsa-miR-524-3p GAAGGCGCUUCCCUUUGGAGU 703 AAGCGCCTT AGCGCCTT GCGCCTT hsa-miR-524-5p CUACAAAGGGAAGCACUUUCUC 704 CCCTTTGTA CCTTTGTA CTTTGTA hsa-miR-525-3p GAAGGCGCUUCCCUUUAGAGCG 705 AAGCGCCTT AGCGCCTT GCGCCTT hsa-miR-525-5p CUCCAGAGGGAUGCACUUUCU 706 CCCTCTGGA CCTCTGGA CTCTGGA hsa-miR-526b CUCUUGAGGGAAGCACUUUCUGU 707 CCCTCAAGA CCTCAAGA CTCAAGA hsa-miR-526b* GAAAGUGCUUCCUUUUAGAGGC 708 AAGCACTTT AGCACTTT GCACTTT hsa-miR-527 CUGCAAAGGGAAGCCCUUUC 709 CCCTTTGCA CCTTTGCA CTTTGCA hsa-miR-532-3p CCUCCCACACCCAAGGCUUGCA 710 GTGTGGGAG TGTGGGAG GTGGGAG hsa-miR-532-5p CAUGCCUUGAGUGUAGGACCGU 711 TCAAGGCAT CAAGGCAT AAGGCAT hsa-miR-539 GGAGAAAUUAUCCUUGGUGUGU 712 TAATTTCTC AATTTCTC ATTTCTC hsa-miR-541 UGGUGGGCACAGAAUCUGGACU 713 GTGCCCACC TGCCCACC GCCCACC hsa-miR-541* AAAGGAUUCUGCUGUCGGUCCCACU 714 AGAATCCTT GAATCCTT AATCCTT hsa-miR-542-3p UGUGACAGAUUGAUAACUGAAA 715 ATCTGTCAC TCTGTCAC CTGTCAC hsa-miR-542-5p UCGGGGAUCAUCAUGUCACGAGA 716 TGATCCCCG GATCCCCG ATCCCCG hsa-miR-543 AAACAUUCGCGGUGCACUUCUU 717 GCGAATGTT CGAATGTT GAATGTT hsa-miR-544 AUUCUGCAUUUUUAGCAAGUUC 718 AATGCAGAA ATGCAGAA TGCAGAA hsa-miR-545 UCAGCAAACAUUUAUUGUGUGC 719 TGTTTGCTG GTTTGCTG TTTGCTG hsa-miR-545* UCAGUAAAUGUUUAUUAGAUGA 720 CATTTACTG ATTTACTG TTTACTG hsa-miR-548a-3p CAAAACUGGCAAUUACUUUUGC 721 GCCAGTTTT CCAGTTTT CAGTTTT hsa-miR-548a-5p AAAAGUAAUUGCGAGUUUUACC 722 AATTACTTT ATTACTTT TTACTTT hsa-miR-548b-3p CAAGAACCUCAGUUGCUUUUGU 723 GAGGTTCTT AGGTTCTT GGTTCTT hsa-miR-548b-5p AAAAGUAAUUGUGGUUUUGGCC 724 AATTACTTT ATTACTTT TTACTTT hsa-miR-548c-3p CAAAAAUCUCAAUUACUUUUGC 725 GAGATTTTT AGATTTTT GATTTTT hsa-miR-548c-5p AAAAGUAAUUGCGGUUUUUGCC 726 AATTACTTT ATTACTTT TTACTTT hsa-miR-548d-3p CAAAAACCACAGUUUCUUUUGC 727 GTGGTTTTT TGGTTTTT GGTTTTT hsa-miR-548d-5p AAAAGUAAUUGUGGUUUUUGCC 728 AATTACTTT ATTACTTT TTACTTT hsa-miR-548e AAAAACUGAGACUACUUUUGCA 729 CTCAGTTTT TCAGTTTT CAGTTTT hsa-miR-548f AAAAACUGUAAUUACUUUU 730 TACAGTTTT ACAGTTTT CAGTTTT hsa-miR-548g AAAACUGUAAUUACUUUUGUAC 731 TTACAGTTT TACAGTTT ACAGTTT hsa-miR-548h AAAAGUAAUCGCGGUUUUUGUC 732 GATTACTTT ATTACTTT TTACTTT hsa-miR-5481 AAAAGUAAUUGCGGAUUUUGCC 733 AATTACTTT ATTACTTT TTACTTT hsa-miR-548j AAAAGUAAUUGCGGUCUUUGGU 734 AATTACTTT ATTACTTT TTACTTT hsa-miR-548k AAAAGUACUUGCGGAUUUUGCU 735 AAGTACTTT AGTACTTT GTACTTT hsa-miR-548l AAAAGUAUUUGCGGGUUUUGUC 736 AAATACTTT AATACTTT ATACTTT hsa-miR-548m CAAAGGUAUUUGUGGUUUUUG 737 AATACCTTT ATACCTTT TACCTTT hsa-miR-548n CAAAAGUAAUUGUGGAUUUUGU 738 ATTACTTTT TTACTTTT TACTTTT hsa-miR-548o CCAAAACUGCAGUUACUUUUGC 739 GCAGTTTTG CAGTTTTG AGTTTTG hsa-miR-548p UAGCAAAAACUGCAGUUACUUU 740 GTTTTTGCT TTTTTGCT TTTTGCT hsa-miR-549 UGACAACUAUGGAUGAGCUCU 741 ATAGTTGTC TAGTTGTC AGTTGTC hsa-miR-550 AGUGCCUGAGGGAGUAAGAGCCC 742 CTCAGGCAC TCAGGCAC CAGGCAC hsa-miR-550* UGUCUUACUCCCUCAGGCACAU 743 GAGTAAGAC AGTAAGAC GTAAGAC hsa-miR-551a GCGACCCACUCUUGGUUUCCA 744 AGTGGGTCG GTGGGTCG TGGGTCG hsa-miR-551b GCGACCCAUACUUGGUUUCAG 745 TATGGGTCG ATGGGTCG TGGGTCG hsa-miR-551b* GAAAUCAAGCGUGGGUGAGACC 746 GCTTGATTT CTTGATTT TTGATTT hsa-miR-552 AACAGGUGACUGGUUAGACAA 747 GTCACCTGT TCACCTGT CACCTGT hsa-miR-553 AAAACGGUGAGAUUUUGUUUU 748 TCACCGTTT CACCGTTT ACCGTTT hsa-miR-554 GCUAGUCCUGACUCAGCCAGU 749 CAGGACTAG AGGACTAG GGACTAG hsa-miR-555 AGGGUAAGCUGAACCUCUGAU 750 AGCTTACCC GCTTACCC CTTACCC hsa-miR-556-3p AUAUUACCAUUAGCUCAUCUUU 751 ATGGTAATA TGGTAATA GGTAATA hsa-miR-556-5p GAUGAGCUCAUUGUAAUAUGAG 752 TGAGCTCAT GAGCTCAT AGCTCAT hsa-miR-557 GUUUGCACGGGUGGGCCUUGUCU 753 CCGTGCAAA CGTGCAAA GTGCAAA hsa-miR-558 UGAGCUGCUGUACCAAAAU 754 CAGCAGCTC AGCAGCTC GCAGCTC hsa-miR-559 UAAAGUAAAUAUGCACCAAAA 755 ATTTACTTT TTTACTTT TTACTTT hsa-miR-561 CAAAGUUUAAGAUCCUUGAAGU 756 TTAAACTTT TAAACTTT AAACTTT hsa-miR-562 AAAGUAGCUGUACCAUUUGC 757 CAGCTACTT AGCTACTT GCTACTT hsa-miR-563 AGGUUGACAUACGUUUCCC 758 ATGTCAACC TGTCAACC GTCAACC hsa-miR-564 AGGCACGGUGUCAGCAGGC 759 CACCGTGCC ACCGTGCC CCGTGCC hsa-miR-566 GGGCGCCUGUGAUCCCAAC 760 ACAGGCGCC CAGGCGCC AGGCGCC hsa-miR-567 AGUAUGUUCUUCCAGGACAGAAC 761 AGAACATAC GAACATAC AACATAC hsa-miR-568 AUGUAUAAAUGUAUACACAC 762 ATTTATACA TTTATACA TTATACA hsa-miR-569 AGUUAAUGAAUCCUGGAAAGU 763 TTCATTAAC TCATTAAC CATTAAC hsa-miR-570 CGAAAACAGCAAUUACCUUUGC 764 GCTGTTTTC CTGTTTTC TGTTTTC hsa-miR-571 UGAGUUGGCCAUCUGAGUGAG 765 GGCCAACTC GCCAACTC CCAACTC hsa-miR-572 GUCCGCUCGGCGGUGGCCCA 766 CCGAGCGGA CGAGCGGA GAGCGGA hsa-miR-573 CUGAAGUGAUGUGUAACUGAUCAG 767 ATCACTTCA TCACTTCA CACTTCA hsa-miR-574-3p CACGCUCAUGCACACACCCACA 768 CATGAGCGT ATGAGCGT TGAGCGT hsa-miR-574-5p UGAGUGUGUGUGUGUGAGUGUGU 769 CACACACTC ACACACTC CACACTC hsa-miR-575 GAGCCAGUUGGACAGGAGC 770 CAACTGGCT AACTGGCT ACTGGCT hsa-miR-576-3p AAGAUGUGGAAAAAUUGGAAUC 771 TCCACATCT CCACATCT CACATCT hsa-miR-576-5p AUUCUAAUUUCUCCACGUCUUU 772 AAATTAGAA AATTAGAA ATTAGAA hsa-miR-577 UAGAUAAAAUAUUGGUACCUG 773 ATTTTATCT TTTTATCT TTTATCT hsa-miR-578 CUUCUUGUGCUCUAGGAUUGU 774 GCACAAGAA CACAAGAA ACAAGAA hsa-miR-579 UUCAUUUGGUAUAAACCGCGAUU 775 ACCAAATGA CCAAATGA CAAATGA hsa-miR-580 UUGAGAAUGAUGAAUCAUUAGG 776 TCATTCTCA CATTCTCA ATTCTCA hsa-miR-581 UCUUGUGUUCUCUAGAUCAGU 777 GAACACAAG AACACAAG ACACAAG hsa-miR-582-3p UAACUGGUUGAACAACUGAACC 778 CAACCAGTT AACCAGTT ACCAGTT hsa-miR-582-5p UUACAGUUGUUCAACCAGUUACU  779 ACAACTGTA CAACTGTA AACTGTA hsa-miR-583 CAAAGAGGAAGGUCCCAUUAC 780 TTCCTCTTT TCCTCTTT CCTCTTT hsa-miR-584 UUAUGGUUUGCCUGGGACUGAG 781 CAAACCATA AAACCATA AACCATA hsa-miR-585 UGGGCGUAUCUGUAUGCUA 782 GATACGCCC ATACGCCC TACGCCC hsa-miR-586 UAUGCAUUGUAUUUUUAGGUCC 783 ACAATGCAT CAATGCAT AATGCAT hsa-miR-587 UUUCCAUAGGUGAUGAGUCAC 784 CCTATGGAA CTATGGAA TATGGAA hsa-miR-588 UUGGCCACAAUGGGUUAGAAC 785 TTGTGGCCA TGTGGCCA GTGGCCA hsa-miR-589 UGAGAACCACGUCUGCUCUGAG 786 GTGGTTCTC TGGTTCTC GGTTCTC hsa-miR-589* UCAGAACAAAUGCCGGUUCCCAGA  787 TTTGTTCTG TTGTTCTG TGTTCTG hsa-miR-590-3p UAAUUUUAUGUAUAAGCUAGU 788 CATAAAATT ATAAAATT TAAAATT hsa-miR-590-5p GAGCUUAUUCAUAAAAGUGCAG 789 GAATAAGCT AATAAGCT ATAAGCT hsa-miR-591 AGACCAUGGGUUCUCAUUGU 790 CCCATGGTC CCATGGTC CATGGTC hsa-miR-592 UUGUGUCAAUAUGCGAUGAUGU 791 ATTGACACA TTGACACA TGACACA hsa-miR-593 UGUCUCUGCUGGGGUUUCU 792 AGCAGAGAC GCAGAGAC CAGAGAC hsa-miR-593* AGGCACCAGCCAGGCAUUGCUCAGC 793 GCTGGTGCC CTGGTGCC TGGTGCC hsa-miR-595 GAAGUGUGCCGUGGUGUGUCU 794 GGCACACTT GCACACTT CACACTT hsa-miR-596 AAGCCUGCCCGGCUCCUCGGG 795 GGGCAGGCT GGCAGGCT GCAGGCT hsa-miR-597 UGUGUCACUCGAUGACCACUGU 796 GAGTGACAC AGTGACAC GTGACAC hsa-miR-598 UACGUCAUCGUUGUCAUCGUCA 797 CGATGACGT GATGACGT ATGACGT hsa-miR-599 GUUGUGUCAGUUUAUCAAAC 798 CTGACACAA TGACACAA GACACAA hsa-miR-600 ACUUACAGACAAGAGCCUUGCUC 799 GTCTGTAAG TCTGTAAG CTGTAAG hsa-miR-601 UGGUCUAGGAUUGUUGGAGGAG 800 TCCTAGACC CCTAGACC CTAGACC hsa-miR-602 GACACGGGCGACAGCUGCGGCCC 801 CGCCCGTGT GCCCGTGT CCCGTGT hsa-miR-603 CACACACUGCAAUUACUUUUGC 802 GCAGTGTGT CAGTGTGT AGTGTGT hsa-miR-604 AGGCUGCGGAAUUCAGGAC 803 TCCGCAGCC CCGCAGCC CGCAGCC hsa-miR-605 UAAAUCCCAUGGUGCCUUCUCCU 804 ATGGGATTT TGGGATTT GGGATTT hsa-miR-606 AAACUACUGAAAAUCAAAGAU 805 TCAGTAGTT CAGTAGTT AGTAGTT hsa-miR-607 GUUCAAAUCCAGAUCUAUAAC 806 GGATTTGAA GATTTGAA ATTTGAA hsa-miR-608 AGGGGUGGUGUUGGGACAGCUCCGU 807 CACCACCCC ACCACCCC CCACCCC hsa-miR-609 AGGGUGUUUCUCUCAUCUCU 808 GAAACACCC AAACACCC AACACCC hsa-miR-610 UGAGCUAAAUGUGUGCUGGGA 809 ATTTAGCTC TTTAGCTC TTAGCTC hsa-miR-611 GCGAGGACCCCUCGGGGUCUGAC 810 GGGTCCTCG GGTCCTCG GTCCTCG hsa-miR-612 GCUGGGCAGGGCUUCUGAGCUCCUU 811 CCTGCCCAG CTGCCCAG TGCCCAG hsa-miR-613 AGGAAUGUUCCUUCUUUGCC 812 GAACATTCC AACATTCC ACATTCC hsa-miR-614 GAACGCCUGUUCUUGCCAGGUGG 813 ACAGGCGTT CAGGCGTT AGGCGTT hsa-miR-615-3p UCCGAGCCUGGGUCUCCCUCUU 814 CAGGCTCGG AGGCTCGG GGCTCGG hsa-miR-615-5p GGGGGUCCCCGGUGCUCGGAUC 815 GGGGACCCC GGGACCCC GGACCCC hsa-miR-616 AGUCAUUGGAGGGUUUGAGCAG 816 TCCAATGAC CCAATGAC CAATGAC hsa-miR-616* ACUCAAAACCCUUCAGUGACUU 817 GGTTTTGAG GTTTTGAG TTTTGAG hsa-miR-617 AGACUUCCCAUUUGAAGGUGGC 818 TGGGAAGTC GGGAAGTC GGAAGTC hsa-miR-618 AAACUCUACUUGUCCUUCUGAGU 819 AGTAGAGTT GTAGAGTT TAGAGTT hsa-miR-619 GACCUGGACAUGUUUGUGCCCAGU 820 TGTCCAGGT GTCCAGGT TCCAGGT hsa-miR-620 AUGGAGAUAGAUAUAGAAAU 821 CTATCTCCA TATCTCCA ATCTCCA hsa-miR-621 GGCUAGCAACAGCGCUUACCU 822 GTTGCTAGC TTGCTAGC TGCTAGC hsa-miR-622 ACAGUCUGCUGAGGUUGGAGC 823 AGCAGACTG GCAGACTG CAGACTG hsa-miR-623 AUCCCUUGCAGGGGCUGUUGGGU 824 TGCAAGGGA GCAAGGGA CAAGGGA hsa-miR-624 CACAAGGUAUUGGUAUUACCU 825 ATACCTTGT TACCTTGT ACCTTGT hsa-miR-624* UAGUACCAGUACCUUGUGUUCA 826 ACTGGTACT CTGGTACT TGGTACT hsa-miR-625 AGGGGGAAAGUUCUAUAGUCC 827 CTTTCCCCC TTTCCCCC TTCCCCC hsa-miR-625* GACUAUAGAACUUUCCCCCUCA 828 TTCTATAGT TCTATAGT CTATAGT hsa-miR-626 AGCUGUCUGAAAAUGUCUU 829 TCAGACAGC CAGACAGC AGACAGC hsa-miR-627 GUGAGUCUCUAAGAAAAGAGGA 830 AGAGACTCA GAGACTCA AGACTCA hsa-miR-628-3p UCUAGUAAGAGUGGCAGUCGA 831 TCTTACTAG CTTACTAG TTACTAG hsa-miR-628-5p AUGCUGACAUAUUUACUAGAGG 832 ATGTCAGCA TGTCAGCA GTCAGCA hsa-miR-629 UGGGUUUACGUUGGGAGAACU 833 CGTAAACCC GTAAACCC TAAACCC hsa-miR-629* GUUCUCCCAACGUAAGCCCAGC 834 TTGGGAGAA TGGGAGAA GGGAGAA hsa-miR-630 AGUAUUCUGUACCAGGGAAGGU 835 ACAGAATAC CAGAATAC AGAATAC hsa-miR-631 AGACCUGGCCCAGACCUCAGC 836 GGCCAGGTC GCCAGGTC CCAGGTC hsa-miR-632 GUGUCUGCUUCCUGUGGGA 837 AAGCAGACA AGCAGACA GCAGACA hsa-miR-633 CUAAUAGUAUCUACCACAAUAAA 838 ATACTATTA TACTATTA ACTATTA hsa-miR-634 AACCAGCACCCCAACUUUGGAC 839 GGTGCTGGT GTGCTGGT TGCTGGT hsa-miR-635 ACUUGGGCACUGAAACAAUGUCC 840 GTGCCCAAG TGCCCAAG GCCCAAG hsa-miR-636 UGUGCUUGCUCGUCCCGCCCGCA 841 AGCAAGCAC GCAAGCAC CAAGCAC hsa-miR-637 ACUGGGGGCUUUCGGGCUCUGCGU 842 AGCCCCCAG GCCCCCAG CCCCCAG hsa-miR-638 AGGGAUCGCGGGCGGGUGGCGGCCU 843 CGCGATCCC GCGATCCC CGATCCC hsa-miR-639 AUCGCUGCGGUUGCGAGCGCUGU 844 CCGCAGCGA CGCAGCGA GCAGCGA hsa-miR-640 AUGAUCCAGGAACCUGCCUCU 845 CCTGGATCA CTGGATCA TGGATCA hsa-miR-641 AAAGACAUAGGAUAGAGUCACCUC  846 CTATGTCTT TATGTCTT ATGTCTT hsa-miR-642 GUCCCUCUCCAAAUGUGUCUUG 847 GGAGAGGGA GAGAGGGA AGAGGGA hsa-miR-643 ACUUGUAUGCUAGCUCAGGUAG 848 GCATACAAG CATACAAG ATACAAG hsa-miR-644 AGUGUGGCUUUCUUAGAGC 849 AAGCCACAC AGCCACAC GCCACAC hsa-miR-645 UCUAGGCUGGUACUGCUGA 850 CCAGCCTAG CAGCCTAG AGCCTAG hsa-miR-646 AAGCAGCUGCCUCUGAGGC 851 GCAGCTGCT CAGCTGCT AGCTGCT hsa-miR-647 GUGGCUGCACUCACUUCCUUC 852 GTGCAGCCA TGCAGCCA GCAGCCA hsa-miR-648 AAGUGUGCAGGGCACUGGU 853 CTGCACACT TGCACACT GCACACT hsa-miR-649 AAACCUGUGUUGUUCAAGAGUC 854 ACACAGGTT CACAGGTT ACAGGTT hsa-miR-650 AGGAGGCAGCGCUCUCAGGAC 855 GCTGCCTCC CTGCCTCC TGCCTCC hsa-miR-651 UUUAGGAUAAGCUUGACUUUUG 856 TTATCCTAA TATCCTAA ATCCTAA hsa-miR-652 AAUGGCGCCACUAGGGUUGUG 857 TGGCGCCAT GGCGCCAT GCGCCAT hsa-miR-653 GUGUUGAAACAAUCUCUACUG 858 GTTTCAACA TTTCAACA TTCAACA hsa-miR-654-3p UAUGUCUGCUGACCAUCACCUU 859 AGCAGACAT GCAGACAT CAGACAT hsa-miR-654-5p UGGUGGGCCGCAGAACAUGUGC 860 CGGCCCACC GGCCCACC GCCCACC hsa-miR-655 AUAAUACAUGGUUAACCUCUUU 861 CATGTATTA ATGTATTA TGTATTA hsa-miR-656 AAUAUUAUACAGUCAACCUCU 862 GTATAATAT TATAATAT ATAATAT hsa-miR-657 GGCAGGUUCUCACCCUCUCUAGG 863 AGAACCTGC GAACCTGC AACCTGC hsa-miR-658 GGCGGAGGGAAGUAGGUCCGUUGGU 864 TCCCTCCGC CCCTCCGC CCTCCGC hsa-miR-659 CUUGGUUCAGGGAGGGUCCCCA 865 CTGAACCAA TGAACCAA GAACCAA hsa-miR-660 UACCCAUUGCAUAUCGGAGUUG 866 GCAATGGGT CAATGGGT AATGGGT hsa-miR-661 UGCCUGGGUCUCUGGCCUGCGCGU 867 GACCCAGGC ACCCAGGC CCCAGGC hsa-miR-662 UCCCACGUUGUGGCCCAGCAG 868 CAACGTGGG AACGTGGG ACGTGGG hsa-miR-663 AGGCGGGGCGCCGCGGGACCGC 869 CGCCCCGCC GCCCCGCC CCCCGCC hsa-miR-663b GGUGGCCCGGCCGUGCCUGAGG 870 CCGGGCCAC CGGGCCAC GGGCCAC hsa-miR-664 UAUUCAUUUAUCCCCAGCCUACA 871 TAAATGAAT AAATGAAT AATGAAT hsa-miR-664* ACUGGCUAGGGAAAAUGAUUGGAU 872 CCTAGCCAG CTAGCCAG TAGCCAG hsa-miR-665 ACCAGGAGGCUGAGGCCCCU 873 GCCTCCTGG CCTCCTGG CTCCTGG hsa-miR-668 UGUCACUCGGCUCGGCCCACUAC 874 CCGAGTGAC CGAGTGAC GAGTGAC hsa-miR-671-3p UCCGGUUCUCAGGGCUCCACC 875 GAGAACCGG AGAACCGG GAACCGG hsa-miR-671-5p AGGAAGCCCUGGAGGGGCUGGAG 876 AGGGCTTCC GGGCTTCC GGCTTCC hsa-miR-675 UGGUGCGGAGAGGGCCCACAGUG 877 CTCCGCACC TCCGCACC CCGCACC hsa-miR-675b CUGUAUGCCCUCACCGCUCA 878 GGGCATACA GGCATACA GCATACA hsa-miR-7 UGGAAGACUAGUGAUUUUGUUGU 879 TAGTCTTCC AGTCTTCC GTCTTCC hsa-miR-7-1* CAACAAAUCACAGUCUGCCAUA 880 TGATTTGTT GATTTGTT ATTTGTT hsa-miR-7-2* CAACAAAUCCCAGUCUACCUAA 881 GGATTTGTT GATTTGTT ATTTGTT hsa-miR-708 AAGGAGCUUACAAUCUAGCUGGG 882 TAAGCTCCT AAGCTCCT AGCTCCT hsa-miR-708* CAACUAGACUGUGAGCUUCUAG 883 AGTCTAGTT GTCTAGTT TCTAGTT hsa-miR-720 UCUCGCUGGGGCCUCCA 884 CCCAGCGAG CCAGCGAG CAGCGAG hsa-miR-744 UGCGGGGCUAGGGCUAACAGCA 885 TAGCCCCGC AGCCCCGC GCCCCGC hsa-miR-744* CUGUUGCCACUAACCUCAACCU 886 GTGGCAACA TGGCAACA GGCAACA hsa-miR-758 UUUGUGACCUGGUCCACUAACC 887 AGGTCACAA GGTCACAA GTCACAA hsa-miR-760 CGGCUCUGGGUCUGUGGGGA 888 CCCAGAGCC CCAGAGCC CAGAGCC hsa-miR-765 UGGAGGAGAAGGAAGGUGAUG 889 TTCTCCTCC TCTCCTCC CTCCTCC hsa-miR-766 ACUCCAGCCCCACAGCCUCAGC 890 GGGCTGGAG GGCTGGAG GCTGGAG hsa-miR-767-3p UCUGCUCAUACCCCAUGGUUUCU 891 TATGAGCAG ATGAGCAG TGAGCAG hsa-miR-767-5p UGCACCAUGGUUGUCUGAGCAUG 892 CCATGGTGC CATGGTGC ATGGTGC hsa-miR-769-3p CUGGGAUCUCCGGGGUCUUGGUU 893 GAGATCCCA AGATCCCA GATCCCA hsa-miR-769-5p UGAGACCUCUGGGUUCUGAGCU 894 AGAGGTCTC GAGGTCTC AGGTCTC hsa-miR-770-5p UCCAGUACCACGUGUCAGGGCCA 895 TGGTACTGG GGTACTGG GTACTGG hsa-miR-802 CAGUAACAAAGAUUCAUCCUUGU 896 TTTGTTACT TTGTTACT TGTTACT hsa-miR-873 GCAGGAACUUGUGAGUCUCCU 897 AAGTTCCTG AGTTCCTG GTTCCTG hsa-miR-874 CUGCCCUGGCCCGAGGGACCGA 898 GCCAGGGCA CCAGGGCA CAGGGCA hsa-miR-875-3p CCUGGAAACACUGAGGUUGUG 899 TGTTTCCAG GTTTCCAG TTTCCAG hsa-miR-875-5p UAUACCUCAGUUUUAUCAGGUG 900 CTGAGGTAT TGAGGTAT GAGGTAT hsa-miR-876-3p UGGUGGUUUACAAAGUAAUUCA 901 TAAACCACC AAACCACC AACCACC hsa-miR-876-5p UGGAUUUCUUUGUGAAUCACCA 902 AAGAAATCC AGAAATCC GAAATCC hsa-miR-877 GUAGAGGAGAUGGCGCAGGG 903 TCTCCTCTA CTCCTCTA TCCTCTA hsa-miR-877* UCCUCUUCUCCCUCCUCCCAG 904 GAGAAGAGG AGAAGAGG GAAGAGG hsa-miR-885-3p AGGCAGCGGGGUGUAGUGGAUA 905 CCCGCTGCC CCGCTGCC CGCTGCC hsa-miR-885-5p UCCAUUACACUACCCUGCCUCU 906 GTGTAATGG TGTAATGG GTAATGG hsa-miR-886-3p CGCGGGUGCUUACUGACCCUU 907 AGCACCCGC GCACCCGC CACCCGC hsa-miR-886-5p CGGGUCGGAGUUAGCUCAAGCGG 908 CTCCGACCC TCCGACCC CCGACCC hsa-miR-887 GUGAACGGGCGCCAUCCCGAGG 909 GCCCGTTCA CCCGTTCA CCGTTCA hsa-miR-888 UACUCAAAAAGCUGUCAGUCA 910 TTTTTGAGT TTTTGAGT TTTGAGT hsa-miR-888* GACUGACACCUCUUUGGGUGAA 911 GGTGTCAGT GTGTCAGT TGTCAGT hsa-miR-889 UUAAUAUCGGACAACCAUUGU 912 CCGATATTA CGATATTA GATATTA hsa-miR-890 UACUUGGAAAGGCAUCAGUUG 913 TTTCCAAGT TTCCAAGT TCCAAGT hsa-miR-891a UGCAACGAACCUGAGCCACUGA 914 GTTCGTTGC TTCGTTGC TCGTTGC hsa-miR-891b UGCAACUUACCUGAGUCAUUGA 915 GTAAGTTGC TAAGTTGC AAGTTGC hsa-miR-892a CACUGUGUCCUUUCUGCGUAG 916 GGACACAGT GACACAGT ACACAGT hsa-miR-892b CACUGGCUCCUUUCUGGGUAGA 917 GGAGCCAGT GAGCCAGT AGCCAGT hsa-miR-9 UCUUUGGUUAUCUAGCUGUAUGA 918 TAACCAAAG AACCAAAG ACCAAAG hsa-miR-9* AUAAAGCUAGAUAACCGAAAGU 919 CTAGCTTTA TAGCTTTA AGCTTTA hsa-miR-920 GGGGAGCUGUGGAAGCAGUA 920 ACAGCTCCC CAGCTCCC AGCTCCC hsa-miR-921 CUAGUGAGGGACAGAACCAGGAUUC 921 CCCTCACTA CCTCACTA CTCACTA hsa-miR-922 GCAGCAGAGAAUAGGACUACGUC 922 TCTCTGCTG CTCTGCTG TCTGCTG hsa-miR-923 GUCAGCGGAGGAAAAGAAACU 923 CTCCGCTGA TCCGCTGA CCGCTGA hsa-miR-924 AGAGUCUUGUGAUGUCUUGC 924 ACAAGACTC CAAGACTC AAGACTC hsa-miR-92a UAUUGCACUUGUCCCGGCCUGU 925 AAGTGCAAT AGTGCAAT GTGCAAT hsa-miR-92a-1* AGGUUGGGAUCGGUUGCAAUGCU 926 ATCCCAACC TCCCAACC CCCAACC hsa-miR-92a-2* GGGUGGGGAUUUGUUGCAUUAC 927 ATCCCCACC TCCCCACC CCCCACC hsa-miR-92b UAUUGCACUCGUCCCGGCCUCC 928 GAGTGCAAT AGTGCAAT GTGCAAT hsa-miR-92b* AGGGACGGGACGCGGUGCAGUG 929 TCCCGTCCC CCCGTCCC CCGTCCC hsa-miR-93 CAAAGUGCUGUUCGUGCAGGUAG 930 CAGCACTTT AGCACTTT GCACTTT hsa-miR-93* ACUGCUGAGCUAGCACUUCCCG 931 GCTCAGCAG CTCAGCAG TCAGCAG hsa-miR-933 UGUGCGCAGGGAGACCUCUCCC 932 CCTGCGCAC CTGCGCAC TGCGCAC hsa-miR-934 UGUCUACUACUGGAGACACUGG 933 GTAGTAGAC TAGTAGAC AGTAGAC hsa-miR-935 CCAGUUACCGCUUCCGCUACCGC 934 CGGTAACTG GGTAACTG GTAACTG hsa-miR-936 ACAGUAGAGGGAGGAAUCGCAG 935 CCTCTACTG CTCTACTG TCTACTG hsa-miR-937 AUCCGCGCUCUGACUCUCUGCC 936 GAGCGCGGA AGCGCGGA GCGCGGA hsa-miR-938 UGCCCUUAAAGGUGAACCCAGU 937 TTTAAGGGC TTAAGGGC TAAGGGC hsa-miR-939 UGGGGAGCUGAGGCUCUGGGGGUG  938 CAGCTCCCC AGCTCCCC GCTCCCC hsa-miR-940 AAGGCAGGGCCCCCGCUCCCC 939 GCCCTGCCT CCCTGCCT CCTGCCT hsa-miR-941 CACCCGGCUGUGUGCACAUGUGC 940 CAGCCGGGT AGCCGGGT GCCGGGT hsa-miR-942 UCUUCUCUGUUUUGGCCAUGUG 941 ACAGAGAAG CAGAGAAG AGAGAAG hsa-miR-943 CUGACUGUUGCCGUCCUCCAG 942 CAACAGTCA AACAGTCA ACAGTCA hsa-miR-944 AAAUUAUUGUACAUCGGAUGAG 943 ACAATAATT CAATAATT AATAATT hsa-miR-95 UUCAACGGGUAUUUAUUGAGCA 944 ACCCGTTGA CCCGTTGA CCGTTGA hsa-miR-96 UUUGGCACUAGCACAUUUUUGCU 945 TAGTGCCAA AGTGCCAA GTGCCAA hsa-miR-96* AAUCAUGUGCAGUGCCAAUAUG 946 GCACATGAT CACATGAT ACATGAT hsa-miR-98 UGAGGUAGUAAGUUGUAUUGUU 947 TACTACCTC ACTACCTC CTACCTC hsa-miR-99a AACCCGUAGAUCCGAUCUUGUG 948 TCTACGGGT CTACGGGT TACGGGT hsa-miR-99a* CAAGCUCGCUUCUAUGGGUCUG 949 AGCGAGCTT GCGAGCTT CGAGCTT hsa-miR-99b CACCCGUAGAACCGACCUUGCG 950 TCTACGGGT CTACGGGT TACGGGT hsa-miR-99b* CAAGCUCGUGUCUGUGGGUCCG 951 CACGAGCTT ACGAGCTT CGAGCTT hsv1-miR-H1 UGGAAGGACGGGAAGUGGAAG 952 CGTCCTTCC GTCCTTCC TCCTTCC hsv1-miR-H2-3p CCUGAGCCAGGGACGAGUGCGACU 953 CTGGCTCAG TGGCTCAG GGCTCAG hsv1-miR-H2-5p UCGCACGCGCCCGGCACAGACU 954 GCGCGTGCG CGCGTGCG GCGTGCG hsv1-miR-H3 CUGGGACUGUGCGGUUGGGA 955 ACAGTCCCA CAGTCCCA AGTCCCA hsv1-miR-H4-3p CUUGCCUGUCUAACUCGCUAGU 956 GACAGGCAA ACAGGCAA CAGGCAA hsv1-miR-H4-5p GGUAGAGUUUGACAGGCAAGCA 957 AAACTCTAC AACTCTAC ACTCTAC hsv1-miR-H5 GUCAGAGAUCCAAACCCUCCGG 958 GATCTCTGA ATCTCTGA TCTCTGA hsv1-miR-H6 CACUUCCCGUCCUUCCAUCCC 959 ACGGGAAGT CGGGAAGT GGGAAGT kshv-miR-K12-1 AUUACAGGAAACUGGGUGUAAGC 960 TTCCTGTAA TCCTGTAA CCTGTAA kshv-miR-K12-10a  UAGUGUUGUCCCCCCGAGUGGC 961 GACAACACT ACAACACT CAACACT kshv-miR-K12-10b  UGGUGUUGUCCCCCCGAGUGGC 962 GACAACACC ACAACACC CAACACC kshv-miR-K12-11 UUAAUGCUUAGCCUGUGUCCGA 963 TAAGCATTA AAGCATTA AGCATTA kshv-miR-K12-12 ACCAGGCCACCAUUCCUCUCCG 964 GTGGCCTGG TGGCCTGG GGCCTGG kshv-miR-K12-2 AACUGUAGUCCGGGUCGAUCUG 965 GACTACAGT ACTACAGT CTACAGT kshv-miR-K12-3 UCACAUUCUGAGGACGGCAGCGA 966 CAGAATGTG AGAATGTG GAATGTG kshv-miR-K12-3* UCGCGGUCACAGAAUGUGACA 967 GTGACCGCG TGACCGCG GACCGCG kshv-miR-K12-4-3p UAGAAUACUGAGGCCUAGCUGA 968 CAGTATTCT AGTATTCT GTATTCT kshv-miR-K12-4-5p AGCUAAACCGCAGUACUCUAGG 969 CGGTTTAGC GGTTTAGC GTTTAGC kshv-miR-K12-5 UAGGAUGCCUGGAACUUGCCGG 970 AGGCATCCT GGCATCCT GCATCCT kshv-miR-K12-6-3p UGAUGGUUUUCGGGCUGUUGAG 971 AAAACCATC AAACCATC AACCATC kshv-miR-K12-6-5p CCAGCAGCACCUAAUCCAUCGG 972 GTGCTGCTG TGCTGCTG GCTGCTG kshv-miR-K12-7 UGAUCCCAUGUUGCUGGCGCU 973 CATGGGATC ATGGGATC TGGGATC kshv-miR-K12-8 UAGGCGCGACUGAGAGAGCACG 974 GTCGCGCCT TCGCGCCT CGCGCCT kshv-miR-K12-9 CUGGGUAUACGCAGCUGCGUAA 975 GTATACCCA TATACCCA ATACCCA kshv-miR-K12-9* ACCCAGCUGCGUAAACCCCGCU 976 GCAGCTGGG CAGCTGGG AGCTGGG 

The invention claimed is:
 1. An oligomer of a contiguous sequence of 7, 8, or 9 nucleotide units in length, for use in reducing the effective amount of a microRNA target in a cell or an organism, wherein at least 70% of the nucleotide units of the oligomer are selected from the group consisting of LNA (Locked Nucleic Acid) units and 2′ substituted nucleotide analogues, and wherein at least 50% of the nucleotide units of the oligomer are LNA units, and wherein at least one of the internucleoside linkages present between the nucleotide units of the contiguous nucleotide sequence is a phosphorothioate internucleoside linkage, wherein the contiguous nucleotide sequence is 100% complementary to the seed sequence of miR-21 (SEQ ID NO: 410), and wherein the seed sequence of miR-21 consists of nucleotides 2 to 8 of SEQ ID NO: 410 counting from the sequence's 5′ end.
 2. The oligomer according to claim 1, for use in the treatment of a medical disorder or disease wherein the medical disease or disorder is glioblastoma, breast cancer, hepatocellular carcinoma, colorectal cancer, sensitization of gliomas to cytotoxic drugs, or cardiac hypertrophy.
 3. A pharmaceutical composition comprising the oligomer according to claim 1, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.
 4. The pharmaceutical composition according to claim 3, further comprising a second independent active ingredient that is a chemotherapeutic agent.
 5. A method for the treatment of a disease or medical disorder associated with the presence or over-expression of a microRNA, comprising the step of administering the pharmaceutical composition according to claim 3 to a patient who is suffering from, or is likely to suffer from said disease or medical disorder.
 6. The oligomer according to claim 1, wherein at least 75% of the internucleoside linkages present between the nucleotide units of the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.
 7. The oligomer according to claim 1, wherein all the internucleoside linkages present between the nucleotide units of the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.
 8. The oligomer according to claim 6, wherein the nucleotide units are selected from the group consisting of DNA, 2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, INA unit, and a 2′-MOE RNA unit.
 9. The oligomer according to claim 6 which comprises a 3′ terminal LNA unit and a 5′ terminal LNA unit.
 10. The oligomer according to claim 6, wherein the length of the oligomer is 7, 8 or 9 contiguous nucleotides, wherein each one of the contiguous nucleotides independently consists of a LNA unit or a 2′ substituted nucleotide analogue unit, and wherein none of the contiguous nucleotides is a DNA unit.
 11. The oligomer according to claim 7, wherein all of the nucleotide units of the contiguous nucleotide sequence are LNA units.
 12. The oligomer according to claim 1, wherein the contiguous nucleotide sequence of the oligomer is 7 nucleotide units in length, all the nucleotide units are LNA units, and all the internucleoside linkages are phosphorothioate.
 13. The oligomer according to claim 1, wherein the contiguous nucleotide sequence of the oligomer is 8 nucleotide units in length, all the nucleotide units are LNA units, and all the internucleoside linkages are phosphorothioate.
 14. The oligomer according to claim 1, wherein the contiguous nucleotide sequence of the oligomer is 9 nucleotide units in length, all the nucleotide units are LNA units, and all the internucleoside linkages are phosphorothioate.
 15. The oligomer according to claim 11, wherein said contiguous nucleotide sequence of the oligomer does not comprise a nucleotide which corresponds to the first nucleotide present in the microRNA sequence of miR-21 (SEQ ID NO: 410) counted from the 5′ end.
 16. The oligomer according to claim 1, wherein the contiguous nucleotide sequence of the oligomer comprises the sequence: (a) 5′-TGATAAGCT-3′ (SEQ ID NO: 9); (b) 5′-GATAAGCT-3′ (SEQ ID NO: 2); or, (c) 5′-ATAAGCT-3′ (SEQ ID NO: 8).
 17. The oligomer according to claim 6, wherein the contiguous nucleotide sequence of the oligomer comprises the sequence: (a) 5′-TGATAAGCT-3′ (SEQ ID NO: 9); (b) 5′-GATAAGCT-3′ (SEQ ID NO: 2); or, (c) 5′-ATAAGCT-3′ (SEQ ID NO: 8).
 18. The oligomer according to claim 7, wherein the contiguous nucleotide sequence of the oligomer comprises the sequence: (a) 5′-TGATAAGCT-3′ (SEQ ID NO: 9); (b) 5′-GATAAGCT-3′ (SEQ ID NO: 2); or, (c) 5′-ATAAGCT-3′ (SEQ ID NO: 8).
 19. The oligomer according to claim 1, wherein the contiguous nucleotide sequence of the oligomer is: (a) 5′-TGATAAGCT-3′ (SEQ ID NO: 9); (b) 5′-GATAAGCT-3′ (SEQ ID NO: 2); or, (c) 5′-ATAAGCT-3′ (SEQ ID NO: 8).
 20. The oligomer according to claim 6, wherein the contiguous nucleotide sequence of the oligomer is: (a) 5′-TGATAAGCT-3′ (SEQ ID NO: 9); (b) 5′-GATAAGCT-3′ (SEQ ID NO: 2); or, (c) 5′-ATAAGCT-3′ (SEQ ID NO: 8).
 21. The oligomer according to claim 7, wherein the contiguous nucleotide sequence of the oligomer is: (a) 5′-TGATAAGCT-3′ (SEQ ID NO: 9); (b) 5′-GATAAGCT-3′ (SEQ ID NO: 2); or, (c) 5′-ATAAGCT-3′ (SEQ ID NO: 8).
 22. The oligomer according to claim 11, wherein the contiguous nucleotide sequence of the oligomer is 7 nucleotide units in length, all the nucleotide units are LNA units, all the internucleoside linkages are phosphorothioate, and all cytosine LNA units are 5-methylcytosines.
 23. The oligomer according to claim 22, wherein the oligomer sequence comprises 5′-ATAAGCT-3′ (SEQ ID NO: 8).
 24. The oligomer according to claim 22, wherein the oligomer sequence is 5′-ATAAGCT-3′ (SEQ ID NO: 8).
 25. The oligomer according to claim 11, wherein the contiguous nucleotide sequence of the oligomer is 8 nucleotide units in length, all the nucleotide units are LNA units, all the internucleoside linkages are phosphorothioate, and all cytosine LNA units are 5-methylcytosines.
 26. The oligomer according to claim 25, wherein the oligomer sequence comprises 5′-GATAAGCT-3′ (SEQ ID NO: 2).
 27. The oligomer according to claim 25, wherein the oligomer sequence is 5′-GATAAGCT-3′ (SEQ ID NO: 2).
 28. The oligomer according to claim 11, wherein the contiguous nucleotide sequence of the oligomer is 9 nucleotide units in length, all the nucleotide units are LNA units, all the internucleoside linkages are phosphorothioate, and all cytosine LNA units are 5-methylcytosines.
 29. The oligomer according to claim 28, wherein the oligomer sequence comprises 5′-TGATAAGCT-3′ (SEQ ID NO: 9).
 30. The oligomer according to claim 28, wherein the oligomer sequence is 5′-TGATAAGCT-3′ (SEQ ID NO: 9).
 31. The method according to claim 5, wherein the disease or medical disorder is cancer.
 32. The oligomer according to claim 1, wherein the oligomer is conjugated with at least one non-nucleotide or non-polynucleotide moiety.
 33. The oligomer according to claim 32, wherein the non-nucleotide or non-polynucleotide moiety is selected from a protein, a fatty acid chain, a sugar residue, a glycoprotein, a polymer, or any combination thereof.
 34. The oligomer according to claim 33, wherein the protein is an antibody.
 35. The oligomer according to claim 33, wherein the polymer is polyethylene glycol.
 36. The oligomer according to claim 1, wherein at least one of the internucleoside linkages present between the nucleotide units of the contiguous nucleotide sequence is not a phosphorothioate or a phosphodiester internucleoside linkage.
 37. The oligomer according to claim 1, wherein at least one cytosine LNA units is not 5-methylcytosine.
 38. The oligomer according to claim 1, wherein at least one cytosine LNA unit is 5-methylcytosine.
 39. The oligomer according to claim 1, wherein all cytosine LNA units are 5-methylcytosines.
 40. The oligomer according to claim 6, wherein at least one cytosine LNA units is not 5-methylcytosine.
 41. The oligomer according to claim 6, wherein at least one cytosine LNA unit is 5-methylcytosine.
 42. The oligomer according to claim 6, wherein all cytosine LNA units are 5-methylcytosines. 